The pancreas derives from cells in the ventral and dorsal foregut endoderm that express the transcription factor Pdx-1. These specified cells give rise to the precursors of the endocrine, ductal, and exocrine pancreatic cells. The identification of transcription factors that regulate the onset of Pdx-1 expression is therefore essential to understand pancreas development. No such factor that acts both in the ventral and in the dorsal endoderm is known. We showed previously that the Onecut transcription factor HNF-6 promotes differentiation of the endocrine cell precursors in which it stimulates expression of the proendocrine gene Ngn-3. By analyzing the phenotype of HNF-6 null mice, we now demonstrate that HNF-6 also controls an earlier step in pancreas development. Indeed, the pancreas of Hnf6(-/-) mice was hypoplastic. This did not result from decreased proliferation or from increased apoptosis, but from retarded pancreatic specification of endodermal cells. The onset of Pdx-1 expression was delayed both in the ventral and in the dorsal endoderm, leading to a reduction in the number of endodermal cells expressing Pdx-1 at the time of pancreatic budding. In normal embryos, HNF-6 was detected in the endoderm prior to the expression of Pdx-1. Moreover, HNF-6 could directly stimulate the Pdx1 promoter. Our data therefore identify HNF-6 as the first factor known to control Pdx-1 expression both in the ventral and in the dorsal endoderm. We conclude that HNF-6 controls the timing of pancreas specification and that HNF-6 acts upstream of Pdx-1 in this developmental process. Together with the known role of HNF-6 in pancreatic endocrine cell differentiation, our data point to HNF-6 as a key regulator of pancreas development.