Three Lions boss Gareth Southgate, the Football Association and Prime Minister Boris Johnson all urged supporters not to boo the anti-racism stance.
But those requests fell on some deaf ears, with an audible round of jeers from some of the expected 22,500 crowd – although cheers from the vast majority soon drowned them out.
The Croatia national anthem was also booed and the reaction followed on from jeers at the two warm-up games played at Middlesbrough’s Riverside Stadium – only this time the whole continent was watching as England fans returned to Wembley for the first time since November 2019.
Southgate said last week that he would not allow his players to continue to be questioned about the knee and the subsequent reaction once the tournament got under way.
A UEFA spokesman said ahead of the game the governing body cannot punish any nations whose supporters do react to the gesture.
"UEFA has a zero tolerance against racism and any player who wants to demand equality amongst human beings by taking the knee will be allowed to do so,” he said.
"We urge spectators to show respect for teams and players taking the knee.
"UEFA will not take disciplinary action against the national association for their fans’ reaction to this initiative as there are no legal grounds to do so in the current competition and disciplinary regulations and to do so would be to punish a national association whose players have taken a stand against racism."