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Human embryonic stem cells (hESCs) depend on glycolysis for energy supply and pluripotency and switch to oxidative phosphorylation upon differentiation. The underlying mechanisms remain unclear. Here, we demonstrate that indoleamine 2,3‐dioxygenase 1 (IDO1) is expressed in primed hESCs and its expression rapidly downregulated upon hESC differentiation. IDO1 is required to maintain pluripotency by suppressing mitochondria activity and promoting glycolysis through the increase of NAD + /NADH ratio. The upregulation of IDO1 during hESC differentiation suppresses the differentiation of hESCs into certain lineages of cells such as cardiomyocytes, which depend on oxidative phosphorylation to satisfy their high energy demand. Therefore, IDO1 plays important roles in maintaining the pluripotency of hESCs.  Stem Cells   2019;37:1158–1165

IDO1 Maintains Pluripotency of Primed Human Embryonic Stem Cells by Promoting Glycolysis