During the course of preimplantation development, the mammalian embryo develops from a single totipotent cell into a blastocyst that is composed of three distinct cell types. Two waves of lineage specification events take place, setting aside a pluripotent cell population, the epiblast, from extraembryonic tissues. The epiblast that will form the somatic cells and germ line of the adult organism remains pluripotent until gastrulation, which commences shortly after the embryo implants. The epiblast's remarkable property of pluripotency has been harnessed by researchers for decades through derivation of embryonic stem cells and epiblast stem cells. Both types of cells can self-renew indefinitely and still retain the ability of germ layer differentiation. However, a central conundrum to the field of stem cell biology is the extent to which these in vitro cultured cells represent their in vivo tissue of origin. In this review we discuss the development of in vivo pluripotency, and compare and contrast the role of signaling pathways and downstream transcription factors in embryo-derived stem cell types and their in vivo equivalent lineage counterparts.