Winks was called up by coach Aidy Boothroyd for England's games against Romania at Wolves' Molineux Stadium on Saturday - which is being dedicated to Cyrille Regis - and a Euro qualifier against Ukraine in Sheffield next Tuesday.
But Winks has now suffered an ankle injury which has resulted in the 22-year-old returning to Tottenham for treatment.
Mason Holgate has also had to withdraw from the squad with an ankle injury.
Winks' call-up to the England U21 squad was his first since missing the 2-0 win away to Ukraine back in November also because of injury.
The midfielder has since played a mostly substitute role for Spurs, with his last appearance coming against Rochdale in the FA Cup in late February.
Boothroyd will now have to do without him and Holgate as he tries to ensure England continue their fine run in Euro qualifying, which has seen the Young Lions win four matches and draw one.