Godolphin's New London had been looming behind O'Brien's Luxembourg in the betting for the marquee Classic on the Epsom Downs but now Ballyodoyle's colt Changingoftheguard is challenging his stablemate after a strong showing on the Roodee.
O'Brien has a cracking record in the Chester Group 3 race with a record nine previous winners of the May Festival contest.
Changingoftheguard set out to make all the running at Chester under Ryan Moore and just when you expected Godolphin's New London to show his gears in the later furlongs, it was Changingoftheguard who found extra pockets of space to run on to a hugely impressive six and a half lengths victory.
Naturally, New London drifted out in the betting while Changingoftheguard was cut for the Classic on June 4 by most firms, meaning O'Brien looks to have the leading hand going into the Derby.
"He was impressive there and we always thought he'd stay well," said O'Brien.
"He was just chinned in his maiden at Punchestown before he then went and won his maiden really well at Dundalk.
"There was no doubt on our part that he would take a step forward from that and improve for going up in trip.
"The one thing we weren't sure about today was the ground when it rained as he's such a fluid mover, but he seems to have handled it fine.
"We were delighted with the run and I think off the back of that, you'd have to say he'd go to Epsom now."
Meanwhile, Alex Merriam, Appleby's assistant had no excuses for New London's disappointing run on the Roodee.
He said: "William (Buick) said he never felt happy.
"We're not blaming the ground or the track, there's not much we can say really.
"This is what trials are all about, we'll see what happens, he was just beaten by a better horse today."
Buick said: "No excuses, he's got a lot to learn."
The Epsom Derby takes place at Epsom racecourse on Saturday, June 04 and starts at 16.30.