Murray State coach Billy Kennedy went with his gut and drew up a play his team had never run before, with Isacc Miles the go-to guy at the end.
Instead, third option Danero Thomas became the hero.
"That shot was bigger than he could imagine," Murray State's B.J. Jenkins said. "It wasn't just a game-winning shot. We'll remember this for the rest of our lives."
For the second time in as many appearances, Vanderbilt (24-9) bowed out early as the higher seed. The Commodores, seeded fourth, were upset by Siena in 2008.
Murray State, the mid-major that came in with a 1-13 all-time NCAA Tournament record, will play Saturday against Butler.
Jermaine Beal made two free throws with 12.7 seconds left to give Vanderbilt the lead. Jenkins missed a rushed three-pointer at the other end, but the ball went out of bounds off a Vanderbilt player with 4.2 seconds left to give Murray State the final shot.
"This is probably the biggest shot I've ever hit in my life," said Thomas, who finished with 11 points, six rebounds, three steals and three blocked shots. "It felt so good, I knew it was going in."
BYU 99, Florida 92
When Jimmer Fredette planted a seed with Florida's Chandler Parsons before their game, he couldn't have known it would sound prophetic.
The BYU star told Parsons, the only Gators player to ever hit two true buzzer-beaters to clinch victories, "no game-winning shots tonight."
Fredette went on to score 37 points and hit two three-pointers in the second overtime to seal the victory in Oklahoma City for the 10th-seeded Cougars (30-4), who reached the second round for the first time in 17 years.
Parsons, meanwhile, missed potential game-winners at the end of regulation and the first overtime, and Fredette didn't let Florida get another chance for a dramatic finish.
"We had a second life, or a third life, or whatever it was, and I just wanted to go out there and try to get it done because I don't know if I could have played another overtime," Fredette said.
Kenny Boynton scored 27 points for the Gators (21-13), helping to rally them from a 13-point second-half deficit in their first NCAA Tournament game since back-to-back national titles in 2006 and 2007.
BYU, which next faces Kansas State, snapped a streak of seven straight first-round losses in the tournament and ended Florida's 12-game NCAA Tournament winning streak.
Kansas St. 82, N. Texas St. 62
On an opening day of upsets and close calls, Kansas State got an early scare.
It lasted 10 minutes.
Denis Clemente had 17 points and six assists for the second-seeded Wildcats (27-7), who gathered themselves after a shaky start in Oklahoma City. The Wildcats held the Mean Green (24-9) to 31 percent shooting and stymied Josh White's forays into the lane, holding North Texas' leading scorer to three points.
Butler 77, UTEP 59
Shelvin Mack hit a career-high seven three-pointers and scored 18 of his 25 points after halftime to lead Butler (29-4) in San Jose. The fifth-seeded Bulldogs extended their best-in-the-nation winning streak to 21.