The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast, in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2-3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as the in vivo-produced embryos. Levels of POU5F1, SOX2, and NANOG mRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired during in vitro culture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related to in vitro culture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species.