The Three Lions entered the match off the back of four straight wins in the section and knowing that another three points would be enough to guarantee their spot at next summer's tournament.
Czech Republic had other ideas, though, and came from behind to record an impressive 2-1 victory - moving level on 12 points with Group A leaders England in the process.
It is the first time that the Three Lions have lost a tournament qualifier for 10 years, and there is now huge pressure on Gareth Southgate's side in their clash away to Bulgaria on Monday night.
The visitors took the lead in just the fifth minute of the contest with Harry Kane converting from the penalty spot, and there was nothing dubious about the decision with Lukas Masopust clearly taking Raheem Sterling's legs from under him as the attacker looked to turn.
It is just the start that the home side would not have wanted as they looked to use the vocal home support to make it an uncomfortable opening to the match for Southgate's side.
The home side did equalise from the resulting corner, though, with Jakub Brabec managing to bundle the ball home from close range after fellow centre-back Ondrej Celustka had made first contact inside the box.
Another set piece caused all sorts of problems inside the England box in the 17th minute, but Patrik Schick headed wide at the far post as the away side managed to survive a dangerous moment.
Czech Republic continued to cause issues in the final third, and Pickford had to be alert to keep out a low strike from Jan Boril in the 24th minute as the pressure continued.
Sterling's pace had brought England's opener and caused a number of problems early on, but the visitors were finding it difficult to keep the ball as the first half continued to gather pace.
Schick headed into the arms of the away side's goalkeeper in the 32nd minute as another opportunity came and went during a period where Southgate's team were just starting to see more of the ball.
Czech Republic had another half-chance in the closing stages of the first period when a quick counter ended with Masopust firing low across goal but there were no takers in a red shirt.
A superior first-half performance saw Jaroslav Silhavy's side have eight attempts to England's four, and there was certainly much for the England boss to ponder at the half-time interval.
The visitors had a huge chance to regain the lead just before the hour when Kane's clever pass released Sterling inside the Czech Republic box, but Sevilla goalkeeper Tomas Vaclik did really well to make contact as the Manchester City attacker looked to round him.
Pickford then had to keep out a curling effort from Masopust down the other end during a period of the match where both teams were having their moments.
Kane's hold-up play was a feature of England's work in the second period, and the Three Lions captain created a chance for Jadon Sancho just past the hour, only for the Borussia Dortmund attacker to be challenged by Brabec at the final moment.
Sterling thought that he had broken into the Czech Republic box in the 67th minute after out-muscling Coufal, but the play was actually stopped for a foul and Sterling booked for dissent.
It was the home side that were looking the most likely, though, and Pickford had to make a smart save at his near post to keep out a powerful strike from Alex Kral 12 minutes from time.
Vaclik then had to make an impressive stop down the other end to prevent Kane from scoring after a fine pass from Barkley had released the Tottenham Hotspur forward.
There was always likely to be a third goal due to the open nature of the contest, and it came for the home side in the 85th minute when FC Dallas forward Zdenek Ondrasek swept a low Masopust cross into the back of the net from close range to register his first international goal.
Tammy Abraham, who has been in red-hot form for Chelsea in the early stages of the 2019-20 campaign, came off the England bench in the 88th minute as Southgate looked for a late response but it never arrived as Czech Republic picked up a deserved three points on home soil.
CZECH REPUBLIC (4-2-3-1): Vaclik; Coufal, Celustka, Brabec, Boril; Kral, Soucek; Jankto (Kopic 82'), Darida, Masopust (Zmrhal 90'); Schick (Ondrasek 65')
ENGLAND (4-3-3): Pickford; Trippier, Keane, Maguire, Rose; Henderson, Rice (Abraham 88'), Mount (Barkley 72'); Sterling, Kane, Sancho (Rashford 72')