It took Kent State two pitchers and 11 pitches to throw a strike in the ninth inning. After a Florida sacrifice bunt, right-hander Josh Pierce hit Daniel Pigott to load the bases.
Pierce once again fell behind, 3-0, to Casey Turgeon, who watched two called strikes on the outside corner before trying unsuccessfully to check his swing.
Justin Shafer flied out to right to end the game, as the Golden Flashes rushed onto the field.
"I don't think I was nervous. More mad and annoyed [at pitchers]," Kent State shortstop Jimmy Rider said of any ninth-inning nerves.
Kent State, which opened the tournament as a No. 3 seed, improves to 47-19 and now faces No. 8 South Carolina at 7 p.m. CT on Wednesday.
The Gamecocks lost, 2-1, to Arkansas in Monday's nightcap.
Florida (47-20) becomes only the second No. 1 national seed to advance to the College World Series and go 0-2, joining Arizona State in 2010.
"It's an unfortunate way to end the season, but I think Kent State deserves a lot of credit," Florida head coach Kevin Sullivan said. "Hung in there, got a big strikeout at the end, and our guys hung in there right to the last out."
Kent State was led by its four stars, three of whom were chosen in the 2012 First-Year Player Draft.
Catcher David Lyon (34th round, Rangers) went 3-for-5, right-hander Ryan Bores (27th round, Rangers) held Florida to two runs in six innings and Rider (26th round, Pirates) had two hits.
First baseman George Roberts went 2-for-4 with two RBIs.
Kent State scored once in the first off Florida starter Hudson Randall, the Tigers' seventh-round Draft pick.
Trainers were called to the mound for Randall in the first, but he finished the inning before leaving because of heat-related issues.
Game-time temperature was 95 degrees, the highest at the College World Series since June 11, 2001.
Florida right-hander Jonathan Crawford started the second and allowed a leadoff single to Jason Bagoly before an error on a sacrifice bunt by third baseman Josh Tobias put two on. After another sac bunt and a strikeout, the Golden Flashes rattled off three straight two-out singles go ahead, 4-0.
"The key was to get runs off Randall, because that bullpen is so good," Stricklin said. "We knew we needed to get some runs."
Florida catcher Mike Zunino -- the No. 3 overall pick by the Mariners -- and Shafer began the comeback by driving in the Gators' first two runs in the third and sixth, respectively.
Florida added two in the seventh off Kent State reliever Brian Clark, who issued a leadoff walk before allowing three straight singles, including an RBI knock by Zunino.
The loss caps another good yet disappointing season for a Florida team that featured nine Draft picks -- eight of whom went in the first 10 rounds -- and spent most of the season as the No. 1 team in the country. It was also Florida's third straight trip to Omaha, none of which have ended with a championship.
"I've been able to make it out here three times, and all three times I've been just as disappointed," Zunino said. "It's one of those things where you don't want it to end. It's going to take a while to hit me. I'm sure it hit the other people. But it's disappointing at the time. I'm sure it will hit harder later."
Kent State 5, Florida 4
Arkansas 2, No. 8 South Carolina 1
No. 2 UCLA (48-15) vs. No. 3 Florida State (49-16), 7 p.m. CT