Background and Aims: HNF4a is a nuclear hormone receptor transcription factor that has been shown to be required for hepatocyte differentiation and development of the liver. It has also been implicated in regulating expression of genes that act in the epithelium of the lower gastrointestinal tract. This implied that HNF4a might be required for development of the gut. Methods: We generated mouse embryos in which HNF4a was ablated in the epithelial cells of the fetal colon using Cre-loxP technology. Embryos were examined using a combination of histology, immunohistochemistry, gene array and RT-PCR, and chromatin immunoprecipitation analyses to define the consequence of loss of HNF4a on colon development. Results: Embryos could be generated until E18.5 that lacked HNF4a in their colon. Although, early stages of colonic development occurred, HNF4a null colons failed to form normal crypts. In addition, goblet cell maturation was perturbed and expression of an array of genes that encode proteins with diverse roles in colon function was disrupted. Several genes whose expression in the colon was dependent on HNF4a contained HNF4abinding sites sequences within putative transcriptional regulatory regions and a subset of these sites were occupied by HNF4a in vivo. Conclusion: HNF4a is a transcription factor that is essential for development of the mammalian colon, regulates goblet cell maturation and is required for expression of genes that control normal colon function and epithelial cell differentiation.