If Italo Calvino has taught me anything, it's that cities are like people. Each has its own distinct characteristics that makes it stand out from the crowd of other cities. Each has a unique history and a confluence of circumstance that proves its past in its very structure. No two cities are completely alike. Thus, when designing cities (whether it be for Gangbusters or for AD&D or something else entirely) it's important to take these things into consideration.
Structure is the first thing to think about. The physical layout of a city, as well as the buildings which comprise it, plays a huge role in what it feels like. Whether the entire place is wattle and daub (High Medieval London) or built of plaster and brick (Ancient Rome) or sheathed completely in marble (Greece) or made of concrete and I-beams (Prohibition New York) is the very first consideration. Cities built of permanent stone are more present, more lasting, and stretch back further into an antique history than those made of temporary materials. Looking up the great stele and temples of a stone city isn't simply looking at the achievements of man, it is peering into the glorious past of that city and seeing the city's fathers. Wooden halls, no matter how glorious, speak only of the present. Stone structures speak of the power of the present extended into the past as a trail of history and extended a well into the future as a promise of solidity.
The physical layout of the city plays into this as well. Cities are like eggs: they start out small, surrounded by a wall, and then shatter their wall and grow larger, leaking out of the "shell." Sometimes a new one is built, but that two is often shattered. At the end stage of their growth they're "scrambled," completely without reference to where their walls once were. Medieval cities tend to be in either stage one (walled), stage two (walled but breached), or stage 2a (walled, breached, re-walled) and to repeat that cycle. However, even when walls are breached they may be retained to form older districts -- and if they aren't, those precincts may still be remembered culturally. For example, the capital of the Third Milean Empire, Miles herself, was once a settlement on a hilltop. This settlement was walled, and still is: but now it is the district known as Pillar Hill, and the gates remain to remind the populace of the powerful imperial presence atop it.
Dividing a city into districts is a good way to get a handle on what may otherwise seem an amorphous mass. These are often known as Quarters in medieval and fantasy sources: the Merchant's Quarter, the Temple Quarter, etc. However, these need not be as cut and dry as the name makes them sound. The Temple Quarter could easily be a long relatively slender portion of the city, representing a few streets where most of the major cults have congregated. The Merchant's Quarter may be a maze of alleys where the mercantile warehouses are located.
Cities also get their character from the physical makeup of roads. In cities of the ancient world, roads were always very wide to allow for processions, which was a key element to the makeup of political power. Processing was an opportunity for classical rulers to see the populace and be seen by them. This means that wide avenues were vital to the political health of ancient cities. However, as the ancient world crumbled and the mechanisms of public power shifted away from the urban centers, the processions fell into disuse. Power resided not in the urbanized population, but in the palaces and the rural settlements in the countryside from which food could be raised. This meant that ordinances providing for wide streets fell into disuse and the great avenues were crazily filled in by new building.
Roads also help determine major trade routes, as do the presence of rivers. Most ancient cities relied on the presence of large bodies of water to connect them to the outside world. Those that didn't had to make do with subsidiary cities that were connected to the water: eg, the Roman docks at Ostia.
Lakes, rivers, and seashore help affect the makeup of cities. Paris has the Seine, Rome has the Tiber, London the Thames. These cities are partially defined by the character of the waterways which they inhabit. This harkens back to the city divisions, as the river is a natural division which easily demarcates different districts or regions.
As far as trade routes go, economy plays a huge factor in the feeling of cities. Ancient or Classical cities, those which serve as major political urban centers because they are cities (Athens, Sparta, Carthage, Tyre, etc.) tended to bend trade to them rather than the other way around. They were wealthy not because they were necessarily well-positioned, but rather because their political and social structure allowed them to become so. An important question indeed is whether a city has become flush with gold because it lies on a trade route... or whether the trade route passes through the city because of its power. The economics of trade can be thought of in a way that is similar to modern descriptions of gravity: a rubber plane over which trade flows, with more and more concentrations of it causing divots, which in turn bends other routes towards those divots. Wealth can accumulate because trade routes cross in a region, but it can also accumulate because a city is mighty and demands tribute, or hauls the riches of conquered nations to its gates.
The populace itself should always be considered: who lives in this place? They need not be the same men who always lived there. In the 9th century, Londonwic was a wooden town built outside the old stone Emporia left behind by Rome. (The question of whether the so-called Anglo-Saxons had completely displaced the native Roman Britons or whether their culture simply changed to be Saxon is beyond the scope of this argument) Cities long dead can be rebuilt by others, or left to crumble.
At the end of the day, great cities should have a character as discernable as any NPC or supporting character, for they are themselves living things comprised of long histories. They are as storied as any individual man, and the scars they bear may last out centuries while their triumphs can be recorded for all time.