Updated project metadata. The Fanconi Anemia (FA) repair pathway governs the repair of highly genotoxic DNA interstrand crosslinks (ICLs) with the assistance of translesion synthesis (TLS), that is facilitated by site-specific monoubiquitination of PCNA (PCNA-Ub) at lysine 164 (K164). Mutation at this residue (K164R) renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair However, the decision-making between the different pathways remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair and pathway preference, we generated a germline Fancg-/- mouse and intercrossed PcnaK164R/+;Fancg-/+ mice. A compound mutation (KR;FGko) was embryonic lethal with the noted exception of very infrequent escapers. RNAseq of primary double mutant mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints, which were rescued by Trp53 knockdown. Upon crosslink induction, KR and FGko MEFs displayed a similar phenotype regarding sensitivity, cell cycle arrest, fork progression and accumulation of single stranded DNA.Remarkably, PCNA-Ub excludes the mismatch recognition complex MSH2/MSH6 from being recruited. Our results implicate a dual function of PCNA-Ub in ICL repair: 1. Facilitate TLS opposite the unhooked ICL and 2. simultaneously exclude MSH2/MSH6 recruitment to channel the ICL towards canonical FA repair.