Myeloid cell leukemia-1 : MCL-1) is a pro-survival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest anti-apoptotic activity. Here we report that depletion of MCL-1, but not its isoform MCL1S, increases genomic instability and cell sensitivity to ionizing radiation (IR) induced death. MCL-1 association with genomic DNA increased post-irradiation and it co-localized with 53BP1 in foci. Post-irradiation, MCL-1 depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR and higher levels of residual 53BP1 and RIF1 foci, suggesting DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells have a reduced frequency of IR-induced BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 co-localization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.