SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.