Windows 10 machines can't connect to 802.1x wireless network authenticating against server using letsencrypt certificate

Might you be running into the issue that Microsoft does "lazy loading" of its roots??

If you go in Microsoft Edge to https://valid-isrgrootx1.letsencrypt.org/, you might find that suddenly ISRG Root X1 has appeared in your root store and things start working better.

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