DNA interstrand crosslinks (ICLs) are repaired by the Fanconi anemia (FA) pathway. The FA pathway is activated by phosphorylation of FANCI in FANCD2-FANCI complex. To investigate how phosphorylation regulates FA pathway activation and function, recombinant FANCD2-FANCI complexes prepared using either the wild-type FANCI or the phosphomimetic FANCI, were comparied using crosslinking/mass spectrometry.