Although not traditional rivals, tensions between the two sides intensified after five years of facing each other in non-League and victory on Saturday secured Luton a league double over Cambridge.
Richard Money, the visitors' manager and former Luton tactician, resisted the urge to make wholesale changes ahead of Wednesday's Old Trafford clash, though his side struggled before the break.
Andrew Drury put Luton ahead in the 19th minute, before the midfielder turned provider for Luke Wilkinson just after the half-hour mark.
Jonathan Smith found the net in first-half stoppage-time to open up a three-goal lead, which proved to be just enough, as Johnny Hunt and Robbie Simpson scored for Cambridge in the second half.
The result leaves Luton four points behind pacesetters Shrewsbury Town, who have played a game more, while Cambridge drop to 14th.
Shrewsbury scraped a 1-0 win at Newport County despite having Keith Southern sent off and Burton Albion stay level on points with them after beating visitors Bury by the same score.
Third-placed Wycombe Wanderers dropped points for the third successive match as they were held to a goalless draw at home to Portsmouth, while Southend United remain on Luton's coattails thanks to Shaquile Coulthirst's late penalty in a 1-0 win over struggling York City.
Also at the wrong end of the table, rock-bottom Hartlepool United gave their survival chances a boost as they beat Plymouth Argyle 3-2, but Mansfield Town were unable to capitalise on York's defeat, as they lost 2-1 at Carlisle United to stay in the bottom two.
Northampton Town – who had Ryan Cresswell dismissed in the 38th minute – became the division's top scorers with a 5-1 triumph at Accrington Stanley.
Cut-price tickets saw a bumper crowd at Dagenham and Redbridge enjoy a 3-1 victory against Cheltenham Town, while Oxford United and Tranmere Rovers also came out victorious, beating Stevenage and Exeter City 2-0 and 2-1 respectively and AFC Wimbledon held Morecambe to a 1-1 draw on the road.
