Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.This research was funded by the Wellcome Trust (091484/Z/10/Z and 095645/Z/11/Z), the Biotechnology and Biological Sciences Research Council (BB/M004023/1 and BB/K010867/1), a European Commission Framework 7 project EuroSyStem (HEALTH-F4-2007-200720 EUROSYSTEM), SysStemCell (ERC-2013-AdG 339431), the Medical Research Council (MRC) (G1100526/1) the Louis-Jeantet Foundation and the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO-VIDI 864.12.007). The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and Medical Research Council (MRC). A.S. is an MRC Professor. Deposited in PMC for immediate release
