Preston boss Simon Grayson has revealed his squad was "devastated" after their loss at Colchester cost them automatic promotion to the Championship.
The Lilywhites were pipped to a top-two spot by MK Dons, despite having begun the final day in second place.
They now face Chesterfield over two legs in the play-off semi-finals, away on Thursday and at Deepdale on Sunday.
"The dressing room is devastated, we're all devastated as staff and the supporters will be," Grayson, 45, said.
He told BBC Radio Lancashire: "We've had a fantastic opportunity to get promoted to the Championship on the last game of the season.
"We knew fate was still in our hands, we knew what we had to do and unfortunately we didn't play as well as we could do."
|Preston's play-off problems...|
|2014: League One play-off semi-final - Rotherham (2-4)||2009: Championship play-off semi-final - Sheffield Utd (1-2)|
|2006: Championship play-off semi-final - Leeds (1-3)||2005: Championship play-off final - West Ham (0-1)|
|2001: First Division play-off final: Bolton (0-3)||1999: Second Division play-off semi-final - Gillingham (1-2)|
|1995: Third Division play-off semi-final: Bury (0-2)||1994: Third Division play-off final - Wycombe (2-4)|
|1989: Third Division play-off semi-final - Port Vale (2-4)|
Grayson has twice been promoted to the Championship through the play-offs with Blackpool and Huddersfield.
Preston - who were on an 18-match unbeaten run in the league before the defeat at Colchester - are the most unsuccessful team in the play-offs since the system was introduced, having failed to win promotion on nine occasions.
"What we've got now is a second opportunity," added Grayson.
"We go on Thursday and Sunday to try and get to Wembley and get promoted that way.
"It's difficult at this moment in time for everybody but we have to go again and psychologically take the players into the play-offs in a positive manner."