Epiblast stem cells (EpiSCs) are isolated from the postimplantation mouse embryo just after implantation but prior to gastrulation. EpiSCs are pluripotent and provide a tractable, in vitro system to study the processes that function during gastrulation to transition pluripotent cells to their differentiated derivatives. This chapter describes the methods for the isolation and maintenance of mouse EpiSCs. We also describe basic assays used to characterize new EpiSC lines.