In the mammalian cecum and colon, a single layer of absorptive, mature enterocytes are a crucial element of the physical barrier to the contents of the lumen. Enterocytic differentiation involves expansion of cytoplasmic cytoskeletal networks, which have been proposed to maintain structural integrity of individual cells and thus the entire epithelial barrier. We sought molecular tools to test this hypothesis in vivo, because in vitro systems displaying full intestinal epithelial differentiation have not yet been developed. Vav proteins are RhoGEFs that modulate cytoskeletal networks in immune cells. We found that Vav proteins were preferentially expressed in terminally differentiating cecal and colonic enterocytes. Loss of Vav protein expression in triple-knockout mice (Vav1(-/-);Vav2(-/-);Vav3(-/-)) resulted in defective expansion of microtubule cytoskeletons, a significant decrease in cell height and diminished expression of differentiation markers. Despite these changes, enterocytes in the triple-mutant mice did not contain measurable alterations in actin cytoskeleton, apical cell-cell junctions, nuclear position or global polarized delivery of proteins involved in terminal differentiation. Aged triple-mutant mice spontaneously developed ulcerative lesions that were, in part, a result of defective wound repair. These studies show that Vav proteins are required for enterocytic differentiation and colonic epithelial barrier integrity.