During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells.