Cryptopatches (CPs) and isolated lymphoid follicles (ILFs) are organized intestinal lymphoid tissues that develop postnatally in mice and include stromal cells expressing the receptor activator of nuclear factor kappa-B ligand (RANKL). We investigated how stromal RANKL influences the development and differentiation of CPs and ILFs by analyzing the development of these lymphoid structures in knockout mice lacking RANKL. We found that RANKL^−/− mice had a fourfold reduction in the overall density of CPs in the small intestine compared to control mice, with the largest decrease in the proximal small intestine. No B cells were present in CPs from the small intestine of RANKL^−/− mice and ILF formation was completely blocked. In sharp contrast, colonic ILFs containing B cells were present in RANKL^−/− mice. Stromal cells within CPs in the small intestine of RANKL^−/− mice did not express CXCL13 (originally called B lymphocyte chemoattractant) and often lacked other normally expressed stromal cell antigens, whereas colonic lymphoid aggregates in RANKL^−/− mice retained stromal CXCL13 expression. The CXCL13-dependent maturation of precursor CPs into ILFs is differentially regulated in the small intestine and colon, with an absolute requirement for RANKL only in the small intestine.