One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis involves the differentiation of inner cell mass cells into primitive and then into parietal endoderm. These processes can be recapitulated in vitro using F9 teratocarcinoma cells, which differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm with subsequent treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on how primitive endoderm develops revealed that the Wnt6 gene is upregulated by RA, leading to the activation of the canonical WNT-β-catenin pathway. The mechanism by which Wnt6 is regulated was not determined, but in silico analysis of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 transcription factors might be involved [1]. Subsequent analysis determined that both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced molecular and morphological markers of primitive endoderm, which occurred concomitantly with the upregulation of the Wnt6 gene. Gata6- or Foxa2-overexpressing cells were also found to have increased levels in T-cell factor (TCF)-dependent transcription, and when these cells were treated with db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and overexpression studies showed that these transcription factors were sufficient to switch on the gene expression of a Wnt6 reporter construct. Together, these results provide evidence for the direct regulation of Wnt6 that leads to the activation of the canonical WNT-β-catenin pathway and subsequent induction of primitive extraembryonic endoderm.