Embryonic stem cells (ESCs) provide an ideal model for investigating developmental processes and are great sources for developing regenerative medicine. Harnessing apoptosis facilitates accurate recapitulation of signalling events during embryogenesis and allows efficient expansion of the ESCs during differentiation. Bcl2, a key regulator of intrinsic anti-apoptotic pathway, encodes two splicing isoforms. However, the identification and functional comparison of Bcl2 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we provide the evidence that both Bcl2 splicing variants are expressed in mESCs. Despite the structural difference, they have similar subcellular localisation. Both Bcl2 alpha and Bcl2 beta enhance differentiation efficiency of the ESCs and effectively improve the survival and growth of ESCs under serum-free conditions. However, the functional effect of Bcl2 alpha was more potent than that of Bcl2 beta. Moreover, only Bcl2 alpha could maintain the long-term expansion and pluripotency of ESCs cultured in serum-free medium. Taken together, our results demonstrate previously unknown functional differences in Bcl2 alternative splicing isoforms in ESCs, and lay the foundation for future efforts to engineer ESCs for regenerative medicine. (C) 2020 The Authors. Published by Elsevier Inc.