Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.