Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.