The shadow England team surged into a 17-point lead with tries from Henry Trinder, Ugo Monye and James Simpson-Daniel but the Barbarians staged a comeback either side of the interval.
Tim Visser, George Smith and Joe Van Niekerk added to the first-half tries from Benoit Baby and Frederic Michalak as the Barbarians took control.
England thought they had escaped with the win with a late try from the Exeter flanker Tom Johnson, but the Barbarians sealed the victory with a second effort from Visser.
Lancaster said: "I was proud of our performance in terms of the inexperience of our side in relation to theirs. We put ourselves in a position at 32-31 where we could have won it.
"I guess at the end their experience and quality told.
"As a lead into the Churchill Cup and for players to get the experience of playing at Twickenham and benchmark themselves against World Cup players, it is valuable."
England team manager Martin Johnson was left frowning in the stands as a handful of World Cup contenders failed to make the most of their chances.
Monye limped off while Simpson-Daniel, having enhanced his credentials with a sparkling try, blotted his copybook by allowing Visser to score his first.
"Ugo has got an MCL (medial collateral ligament) strain. He will be assessed in the morning but it doesn't look too while Matt Banahan has a soft tissue injury," said Lancaster.
"(Joe) Marler has a mild concussion. It wasn't spinal. That is the protocol to get him off field."
On the positive side, captain Luke Narraway had a hand in the tries scored by Trinder and Simpson-Daniel and will be pressing for a place in England's World Cup training squad.
Banahan enjoyed a confident trial run at inside centre, his different third position in an England jersey this season, and he seems certain to travel to New Zealand as a utility back.
"I tried to play to the best of my ability," said Narraway.
"If it is enough to make the World Cup training squad I'll be happy.
"This was a massive massive opportunity because some of those guys were going out there for the first time against world class opposition."
The England Saxons squad for the Churchill Cup will be named on Monday and is set to include a red rose return for Saracens prop Matt Stevens.
Jordan Crane and Andy Saull will be checked over after sustaining injuries in the Aviva Premiership while the likes of Banahan and Charlie Hodgson have been told to rest ahead of the World Cup build-up.
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