Summary: Human pluripotent stem cells (hPSCs) maintain a highly fragmented mitochondrial network, but the mechanisms regulating this phenotype remain unknown. Here, we describe a non-cell death function of the anti-apoptotic protein, MCL-1, in regulating mitochondrial dynamics and promoting pluripotency of stem cells. MCL-1 is induced upon reprogramming, and its inhibition or knockdown induces dramatic changes to the mitochondrial network as well as loss of the key pluripotency transcription factors, NANOG and OCT4. Aside from localizing at the outer mitochondrial membrane like other BCL-2 family members, MCL-1 is unique in that it also resides at the mitochondrial matrix in pluripotent stem cells. Mechanistically, we find MCL-1 to interact with DRP-1 and OPA1, two GTPases responsible for remodeling the mitochondrial network. Depletion of MCL-1 compromised the levels and activity of these key regulators of mitochondrial dynamics. Our findings uncover an unexpected, non-apoptotic function for MCL-1 in the maintenance of mitochondrial structure and stemness. : Gama and colleagues show that MCL-1 regulates mitochondrial network morphology in human pluripotent stem cells. MCL-1 downregulation resulted in loss of OCT4 and NANOG and an elongated mitochondrial network. MCL-1 associates with mitochondrial dynamics regulators; this association is disrupted by an MCL-1 small-molecule inhibitor. The results provide mechanistic insight into the connection between apoptosis, mitochondrial dynamics, and pluripotency. Keywords: apoptosis, MCL-1, pluripotent stem cells, mitochondria, DRP-1, OPA1, mitochondrial dynamics, pluripotency