The drive: From Rochester NY to Albany NY along the NYS Thruway. The Rochester airport is at an altitude of 559 ft MSL, the Albany airport 285. Prevailing winds are out of the west.
The car: 2010 Ford Fusion V6 AWD.
The bet: Will my gas mileage differ significantly driving from Rochester to Albany (net downhill, with the wind) compared with Albany to Rochester (net uphill, against the wind) and if so, how much?
It’s not a critical question, of course. I thought I would test the combined wisdom of the board against real life 
Wind would be bigger effect, both add together, so it should be better with wind/downhill. How much? Who knows!
I suspect there are too many variables to think that any changes in mileage are attributable to the altitude and wind variables that you describe.
Wind and altitude changes do make a difference, but it needs to be a controlled experiment to be able to accurately measure their effects on mileage.
The 2010 Fusion weighs (WAG guess) 3500#.
The altitude difference is (559-285) is 274’.
3500*274=959,000 ft.lb
Given that 1HP=550ft.lb/sec, the altitude difference requires 1743HP.secs of work (basically, 1HP output for 1/2 hr.)
Given that a decent engine will require 1 gallon of gas to output 20 hp for 1 hr, the increased usage of gas due solely to altitude ought to be roughly 1/40 gal=3.2 fl. oz. Given that the fuel burn for the trip ought to be (another WAG) 8 gal gas, the altitude change ought to vary fuel burn by 0.3%.
(Please note that this is purely academic, and in any event is moot if one doesn’t stop/start at the airports: airports are generally on the highest land around, and thus are only roughly representative of the town they serve.)
Feel free to cut me in on the winnings 
Are you driving behind another vehicle?
Define “significantly”.
This is not rocket science.
If you can be assured of a constant wind then yes you’ll probably see an increase in gas mileage…but I travel a good portion of that section of the tpk several times a year…Many times the prevailing wind is coming from the North East…Just too many factors…Now if you travel that all the time…Then yes over time you’re gas mileage should be slightly higher going from Rochester to Albany…But taking a bet on just ONE trip…I don’t know.
Too many other variables, i.e. speed, acceleration, idling time, temperature, etc, to base any mileage difference you see solely on the wind/altitude combination.
Need to know if you use EZPass or not? What exits?
EZ-Pass or NOT wouldn’t make a difference on the NY-State Turnpike.
Assuming that speed, traffic conditions, and weather remain constant, the difference in altitude will NOT make a significant difference. Without having checked MeanJoe’s figures, a factor of .003 sounds good to me. The wind might, but we don’t know how much wind we’re talking here.
Oh, and the purpose of the trip…are you picking up your Mother-In-Law?
It all depend on if you accelerate quickly or slowly come up to speed:) or it you draft a truck,or if your tires are rocks or marshmellows or if, or if, or if…
All things equal, I don’t think there will enough of a difference for you to ever detect.
For what it’s worth, I live at a shade over a 1000 feet altitude and visit CO on a regular basis with altitudes varying from 5 to 10k feet. The mileage in my Lincoln actually improves going up there as opposed to the trip back which is a gradual downhill.