Gov. Ron DeSantis on Friday revoked Disney’s special tax privileges in Florida, culminating an extraordinary clash between one of the Republican Party’s leading figures and a powerful company with deep historical ties in his state.

The move, which reverses a 55-year arrangement allowing the company to self-govern its theme park complex, came after a weekslong battle with Disney that became a symbol of the country’s broader cultural fights over education, sexuality and identity.

The Florida standoff largely centered on an education law recently signed by Mr. DeSantis. That law, called the “Parental Rights in Education” measure — or, to its critics, “Don’t Say Gay” — prohibits classroom instruction about sexual orientation and gender identity in some elementary school grades. After internal demands to speak out, Disney, Florida’s largest private employer, had criticized the measure and paused political donations.

“I’m just not comfortable having that type of agenda get special treatment in my state,” Mr. DeSantis said on Friday, denouncing how Disney had responded to the education legislation as well as videos circulated in the conservative media that showed employees at the company discussing matters of sexuality. A representative for Disney declined to comment on Mr. DeSantis’s signing of the measure.

Widely seen as retaliation, Mr. DeSantis’s move illustrated just how drastically the G.O.P. has transformed from the days when its leaders often moved in lock step with the nation’s largest businesses. The episode also showed how major companies have felt rising pressure to take a stand on heated political issues.

Over the last decade or so, and especially after former President Donald J. Trump’s ascent, leading Republicans have increasingly seen political benefit in criticizing corporate America.

“Republicans who for a long time have had a close and warm relationship with U.S. corporations have started to be more selective about those relationships,” said former Representative Carlos Curbelo, a Florida Republican, adding that the party’s lawmakers were not “afraid to confront even some popular brands that before would have been unthinkable.”

He went on, “Whereas before it would have been unimaginable that a politician, especially a Florida politician, would confront Mickey Mouse, now there’s actually significant political incentive to do so.”

Mr. DeSantis, a possible 2024 presidential contender, has been at the forefront of national battles over curriculums, classrooms and sports teams, issues that have galvanized many conservative voters.

Democrats and advocacy organizations have been sharply critical of conservative efforts on those topics, and a handful of Republican lawmakers around the country have questioned whether the flurry of legislative activity on these subjects is aimed at real-world problems.

L.G.B.T.Q. leaders and some education officials have forcefully criticized the education law that set off the dispute in Florida, saying that it is already having a chilling effect on teachers and may do the same for young students who have relied on schools as a safe place to talk about personal issues.

“Governor DeSantis is wielding the power of the state to punish businesses simply for not falling in line with his brutal and discriminatory attacks,” said Joni Madison, the interim president of the Human Rights Campaign, an L.G.B.T.Q. advocacy group. “Every step of this entire ordeal in Florida has been nothing more than a shameful attempt to out-Trump Trump through fear, hate and lies.”

But for Republican politicians, the decision to take on a major company over the issue is less likely to backfire with today’s conservative base than in an earlier era when business-minded moderates had far more influence in the party.

“This is, in my view, a low-risk engagement here against a company that is perceived by many Republican base voters to be aggressively pursuing or promoting a policy agenda,” Mr. Curbelo said.

That shift, evident with the rise of the Tea Party during the Obama administration, plainly intensified amid the political realignment of the Trump era, as Republicans increasingly became the party of white working-class voters while some big-business and tech leaders — and many of their employees — moved left.

At the bill signing on Friday, where Mr. DeSantis also approved measures including the so-called Stop W.O.K.E. Act, which restricts how race is discussed in schools and businesses, the governor blasted not only Disney but also Google and Coca-Cola for what he cast as diversity measures that were overly ideological.

At the mention of Disney, the Florida crowd booed.

Patricia Mazzei contributed reporting.