NANOG, one of homeobox proteins, plays a crucial role in regulating self-renewal and pluripotency for embryonic stem cells (ESCs). Since the ubiquitin-mediated degradation of NANOG protein has been implicated in its cellular functions involved in not only maintenance of pluripotency and pluripotent epiblast, but also prevention of primitive endoderm differentiation, the identification of ubiquitin ligases and deubiquitinating enzymes (DUBs) for NANOG is required to elucidate its protein stability and the regulation of cellular functions in these processes. In this study, we have identified a novel deubiquitinating enzyme USP21 which interacts with NANOG by both yeast two hybrid screening for DUBs and immunoprecipitation analyses. These analyses revealed that USP21 specifically regulates the Lys48-linked polyubiquitination and stability of NANOG, providing a new way of maintaining the pluripotency of ESCs and induced pluripotent stem cells (iPSCs). (C) 2016 Elsevier Inc. All rights reserved.