Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre–implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency, the transcription factor Oct–4, in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct–4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct–4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059, which suppresses activation of the mitogen–activated protein kinases Erk1 and Erk2, results in increased persistence of Oct–4–expressing cells. Oct–4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline–competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time–window. Procedures that extend this time–window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation.