Yes, lead times times for transport connections is likely a factor in many "village" stays but how big a factor that is may depend on the goal: lazing around most of the time or doing a fair amount day trips to the listed cities. If it's the latter, I'd definitely choose to stay IN those cities.
Florence is one of those terrific hubs for day tripping...except 90 minutes to Rome is too far for me, personally, for taking multiple trips there. It's a better hub for Lucca, Pisa, Siena, Fiesole, etc. The other problem here is that Florence isn't on the sightseeing wish list to begin with (and I'm sort of curious why not)?
Anyway, as suggested by , it's most efficient to split your time if quality city sightseeing is in the plan. If I was to spend multiple days out of a two-week stretch sightseeing Rome, I'd stay in Rome; there's just so much ground to cover in that one. Venice? I'd stay those days in Venice. Milan? Maybe two weeks in Varenna: an hour by train to the city, and ferry service to towns around the lake. Milan tends to be the least interesting to tourists among the three listed so having that option is a bonus, IMHO.
"Ambiance" isn't restricted to small towns; it's alive and well in some of the cities too. :O)
Kyle, you haven't said what time of year you're planning to go?