Ablation of the mouse genes for Onecut-2 and Onecut-3 was reported previously, but characterization of the resulting knockout mice was focused on in utero development, principally embryonic development of liver and pancreas. Here we examined postnatal development of these Onecut knockout mice, especially the critical period before weaning. Onecut-3 knockout mice develop normally during this period. However, Onecut-2 knockout mice fail to thrive, lagging behind their littermates in size and weight. By postnatal day (d)19, they are consistently 25–30% smaller. Onecut-2 knockout mice also have a much higher level of mortality before weaning, with only ∼70% survival. Interestingly, Onecut-2 knockout mice that are heterozygous for the Onecut-3 knockout allele are diminished even further in their ability to thrive. They are ∼50–60% as large as their normal-sized littermates at d19, and less than half of these mice survive to weaning. As reported previously, the Onecut-2/Onecut-3 double knockout is a perinatal lethal. Microarray technology was used to determine the effect of Onecut-2 ablation on gene expression in duodenum, whose epithelium has among the highest levels of Onecut-2. A subset of intestinally expressed genes showed dramatically altered patterns of expression. Many of these genes encode proteins associated with the epithelial membrane, including many involved in transport and metabolism. Previously, we reported that Onecut-2 was critical to temporal regulation of the adenosine deaminase gene in duodenum. Many of the genes with altered patterns of expression in Onecut-2 knockout mouse duodenum displayed changes in the timing of gene expression.