The first two cell lineages of the mammalian embryo - the inner cell mass(ICM) and the trophectoderm - segregate at the blastocyst stage. While the transcription factors Oct4 and Nanog positively regulate commitment to the ICM lineage, the positive regulators of trophectoderm cell fate remain unknown. Strumpf and colleagues now report that the homeodomain protein Cdx2 and the T-box protein Eomes are required at distinct stages during trophectoderm lineage development in mice (see p. 2093). Their studies of homozygous mutant embryos reveal that Eomes is required for trophectoderm differentiation and proliferation beyond the expanded blastocyst stage, while Cdx2 is essential for trophectoderm function, for establishment of the trophoblast lineage, and for restriction of Oct4and Nanog expression to the ICM lineage. Thus, Cdx2 is the earliest known transcription factor involved in trophectoderm specification and is essential for the segregation of the ICM and trophectoderm lineages.