The histone acetyltransferase Myst2 plays an important role in embryogenesis, but its function in undifferentiated ES cells remains poorly understood. Here, we show that Myst2 plays a role in pluripotency and self‐renewal of ES cells. Myst2 deficiency results in loss of characteristic morphology, decreased alkaline phosphatase staining and reduced histone acetylation, as well as aberrant expression of pluripotency and differentiation markers. Our ChIP data reveal a direct association of Myst2 with the Nanog promoter and Myst2‐dependent Oct4 binding on the Nanog promoter. Together our data suggest that Myst2‐mediated histone acetylation may be required for recruitment of Oct4 to the Nanog promoter, thereby regulating Nanog transcription in ES cells.