MK's 6-1 win over the non-league side means that following AFC's victory over York on Monday night, the two will go against each other at the start of December in Buckinghamshire in what will no doubt be an emotion-fuelled affair.
AFC were formed in 2002 by supporters of Wimbledon FC who were unhappy with the Football Association's decision to allow their club to relocate to Milton Keynes.
A campaign for Milton Keynes to drop the 'Dons' part of their name is ongoing, and the Wimbledon Independent Supporters Association has expressed a desire for the FA Cup tie not to be played. They have promised to speak with the club and its supporters about whether or not it should be.
After their victory over York on Monday night, AFC manager, Neal Ardley, a former Wimbledon FC player, said: "I'd play against anyone."
On Tuesday, two goals from Shaun Williams combined with efforts from James O'Shea, Dean Bowditch, Dele Ali and Adam Chicksen to set up the second-round clash at stadium:mk; Dave Theobald netted City's consolation.
Elsewhere in the FA Cup on Tuesday, League One leaders Tranmere won 3-0 at non-league Braintree Town in their delayed first-round tie. Teenager Cole Stockton scored, in between creating goals for Joe Thompson and Max Power. Rovers will host Chesterfield in round two.
Gary McDonald's goal on the stroke of half-time saw Morecambe win 1-0 away to League Two rivals Rochdale. The Shrimps' reward is a trip to Coventry's Ricoh Arena.
A thriller at Valley Parade saw Bradford progress on penalties against Northampton - the Bantams' eighth success in a row in shoot-outs. The hosts won 4-2 on spot-kicks after a 3-3 draw and will host Brentford next.
Will Atkinson and Ishmel Demontagnac (penalty) traded first-half goals, and Nahki Wells' spot-kick looked to have won the tie before Clive Platt levelled at the death. Kelvin Langmead put the visitors in front in extra-time, only for Carl McHugh to equalise in the 120th minute - and Ricky Ravenhill's penalty proved decisive in the shoot-out.
Walsall crashed out 3-2 after extra-time at home to Lincoln City. Vadaine Oliver was the hero for the Blue Square Premier battlers, coming off the bench to score twice in extra-time. Alan Power and the Saddlers' Richard Taundry scored in normal time, with Jamie Paterson's free-kick proving no more than a consolation.
The Imps will be at home to Mansfield, who won 4-1 on penalties at Slough Town after the tie finished 1-1 in 120 minutes.
Garry Hunter scored in stoppage time as Barrow won 1-0 at home to Guiseley to ensure they will entertain Macclesfield.
Scott Rendell's brace enabled Luton to win 2-0 at Nuneaton, setting up a tie against Dorchester Town at Kenilworth Road.