The cytomegalic form of adrenal hypoplasia congenita is an X-linked disease caused by mutations in NR0B1, which codes for Dax1. Failure to create a conventional knock-out mouse model led us to hypothesize that Dax1 is expressed earlier than the onset of steroidogenesis at embryonic day 9 (E9) and with a novel function independent of steroidogenesis. Previously, we reported Dax1 expression in ES cells and preimplantation embryos. Our purpose here was to identify Dax1 expression after implantation and to examine its expression patterns throughout embryonic development. We used immunohistochemistry (IHC) and western blot analysis to determine Dax1 protein expression in murine embryos from E5 through E11. IHC was also used to investigate the expression of the following proteins involved in steroidogenesis: progesterone, estrogen (α and β) and androgen receptors; Wilms tumor 1 (Wt1); steroidogenic factor 1(Sf1); and cytochrome P450scc. Dax1 expression was observed at a critical boundary throughout embryonic development; specifically, it was expressed in all three layers (endoderm, mesoderm, and ectoderm) of the embryo proper, and it distinguished between the embryo and its extra-embryonic endoderm (ExE), where it was not expressed. In contrast, Sf1 and Wt1 were found to be expressed throughout the embryo proper as well as the ExE. The progesterone, estrogen (α and β) and androgen receptors, and cytochrome P450scc were not observed to be expressed in extra-embryonic or embryonic cell layers, as compared to positive control tissues. Western blot analysis confirmed the IHC results. Based on the expression patterns observed in embryonic development, we hypothesize that Dax1 and its network partners, Wt1 and Sf1, are critical in early embryonic development and play roles different from those in steroidogenesis.