The Commission said it had sent a formal request for information to X in what is a first procedure launched under Brussels' new European Digital Services Act (DSA). It comes two days after it fired off a warning letter from internal market Commissioner Thierry Breton.
In a statement, the Commission said it was responding to "indications received concerning the presumed transmission of illicit content."
Its demand for clarification comprises a 40-page document with a raft of specific questions.
Twitter has until October 18 to respond, with a deadline of October 31 for less urgent aspects of the demand for information.
Breton, the Commission's self-styled "digital enforcer," told AFP that Thursday's move is about "protecting our citizens and democracies in offering users a safe environment and reliable sources of information -- including in times of crisis."
X has defended itself against earlier claims from the European Union that it is failing to tackle disinformation around the Gaza-Israel conflict as Brussels investigates.
The firm's CEO Linda Yaccarino wrote that the platform, formerly Twitter, had "taken action to remove or label tens of thousands of pieces of content" and removed hundreds of accounts linked to Gaza militant organisation Hamas, which attacked Israel on Saturday.
She addressed the letter, dated Wednesday, to Breton, who traded barbs with Musk on social media after accusing the platform of allowing "violent and terrorist content" to circulate.
Breton has sent similar letters of alarm to Mark Zuckerberg, boss of Facebook parent Meta, and on Thursday to TikTok and its CEO Shou Zi Chew.
In each case, Breton gave the platforms 24 hours to get back to him with details of what they are doing to crack down on "illegal content and disinformation" reportedly circulating in posts.
Breton stressed that the large online platforms are now subject to the EU's Digital Services Act (DSA), legislation that came in two months ago that requires them to crack down on content deemed illegal under EU law or laws of individual EU countries.
Violations of the DSA can be met with mandatory remedial measures to halt such content, or fines that could go up to six percent of a company's global turnover, or potentially even steps to ban offending platforms from Europe.
Breton posted copies of each of his letters to his accounts on X and on an X rival called Bluesky.
X is especially fixed in Brussels' crosshairs because Musk, who bought Twitter for $44 billion last year, has gutted its staff, including content moderators, in a bid to save money.
- Young TikTok users at risk -
Yaccarino's response letter to Breton, reposted by the CEO on her X account, said the firm had taken down posts that involved "violent speech, manipulated media and graphic media".
She said that more than 700 notes were added to posts in the first four days after the violence erupted in Israel, and they were seen tens of millions of times.
In his letter to TikTok, Breton stressed that its users, who are mainly young, were especially vulnerable to fake and manipulated information.
"Given that your platform is extensively used by children and teenagers, you have a particular obligation to protect them from violent content depicting hostage taking and other graphic videos which are reportedly widely circulating on your platform, without appropriate safeguards," Breton said.
To Zuckerberg, Breton noted that Meta had made some efforts at content moderation but urged it to be "vigilant" about meeting DSA requirements in light of the current Israel-Hamas conflict.
A Meta spokesperson said in reply the company had quickly set up monitoring teams with experts speaking Hebrew and Arabic, who were working with fact checkers to curb disinformation, and that "we'll continue this work as this conflict unfolds".
AFP fact-checkers have found several posts on X, Facebook and TikTok promoting a fake White House document allocating $8 billion in military assistance to Israel.
And several platforms have had users passing off material from other conflicts, or even from video games, as footage from Israel or Gaza.
The EU recently rated X as the worst of any major platform for illegal online content based on a pilot analysis, and Musk has pulled out of a voluntary EU code of practice on battling disinformation.
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