The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1) and sucrase-isomaltase (SI) are well characterized markers of these transitions. We have previously shown in mature jejunum using gene inactivation models that Gata4 and Hnf1 are each indispensable for LPH and Fabp1 gene expression, but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1 regulate LPH, Fabp1 and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1 resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1 had a partial effect (~50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling, but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1 protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of mRNA. Finally, we show that neither Gata4 nor Hnf1 mediates the glucocorticoid-induced precocious maturation of the intestine, but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1 that are dependent on the developmental time-frame in which they are expressed.