Czech Republic scored two late goals to dump England out of the European Under-21 Championship.
England needed to win to progress to the last four and looked on track to achieve that after Danny Welbeck's 76th-minute header gave England the lead.
But substitutes Jan Chramosta and Tomas Pekhart tapped home in the 89th minute and stoppage time respectively.
Czech Republic will now face Switzerland in Wednesday's semi-final.
Stuart Pearce reacted to his side's stalemate with Ukraine by dropping Jack Rodwell, Danny Rose and captain Michael Mancienne.
With Manchester United's new centre-back Phil Jones handed the armband, Tom Cleverley reinstated to the starting line-up and Fabrice Muamba and Scott Sinclair brought in, there was a new look to the England team.
The lively Daniel Sturridge created the first opening in the 10th minute, picking out Welbeck, who peeled off his marker on the edge of the box but could only fire straight at Czech goalkeeper Tomas Vaclik.
Vaclik's opposite number, Frankie Fielding, was then forced into a reflex save from Lukas Marecek, whose well improvised volley from the edge of the box provided a moment of concern for Pearce's side.
That opening ushered in a good spell for the Czechs, as England's early dynamism subsided.
But England again came close in the 27th minute, with a patient and confident build-up resulting in Ryan Bertrand's chipped cross from the left that reached Cleverley at the far post, only for him to hack his effort into the side netting.
Sinclair, whose introduction on the left seemed to bring the best out of Bertrand, came close four minutes before half-time when his surging run infield from the left had the Czechs stretched, before his thunderous shot was tipped over the bar by Vaclik.
England were guilty of surrendering possession cheaply throughout, with Jordan Henderson central to a midfield that lacked guile or imagination against mediocre opposition.
It was no surprise when Liverpool's latest signing was replaced by Henri Lansbury halfway through the second half - and the substitute made an immediate impact with a clever reverse pass to Sinclair, whose low cross from the left had the Czechs stretched before they eventually cleared.
England eventually breached the Czechs' last line of defence with 14 minutes left and it was no surprise to see Sturridge involved once again. His teasing cross from the right was met by the head of Welbeck, and the relief was tangible as England appeared to be heading for the last four.
But Pearce's team conceded a cruel late equaliser, when Chramosta came off the bench to poke home a scrappy finish after England had failed to clear.
To add insult to injury, Chramosta took advantage of the space at the back as England pushed for an even later winner, teeing up former Spurs striker Pekhart to stroke home a second for Czech Republic.
In the absence of Jack Wilshere and Andy Carroll, England lacked creativity and a genuine goal threat throughout - a problem which has ultimately resulted in their failure to reach the semi-finals.
England Under-21 coach Stuart Pearce:
"We are disappointed as a group as we felt after taking the lead we should have held on to it.
"They didn't really cause us a problem all evening defensively but we switched off for a minute, it falls to their player, and it's 1-1. The second goal was neither here nor there.
"It was probably the best performance of the tournament. But perhaps us going home now was symptomatic of us not passing the ball well enough in the first two games.
"This is a culmination of a two-year period, not just a one-off. The players put their hearts and souls into it. And for one or two it's the end of the line at Under-21 level."