Anterior visceral endoderm (AVE) plays essential roles with respect to anterior-posterior axis development in the early mouse embryo. To assess the genetic cascade involved in AVE formation, the cis-regulatory elements directing expression of vertebrate Otx2 genes in the AVE were analyzed via generation of transgenic mice. Otx2 expression in AVE is regulated directly by the forkhead transcription factor, Foxa2. Moreover, Foxa2 is essential for expression of the Wnt antagonists, Dkk1 and Cerl, in visceral endoderm during the pre- to early streak stages; however, Foxa2 appears to be dispensable for subsequent Dkk1 expression associated with forebrain induction. Thus, we propose that Foxa2 is crucial in early anterior-posterior axis polarization in terms of regulation of expression of AVE-specific genes. These findings provide profound insights into conserved roles of Foxa2 transcription factors in anterior specification throughout the evolution of the chordate body plan.