Lymph node (LN) organogenesis is initiated by the interaction between hematopoietic lymphoid tissue inducer (LTi) cells and the mesenchymal organizer cells. Mice in which the IL-7 signaling pathway has been disrupted have a severe defect in LN development; however, the reasons underlying this defect are as yet unknown. In this study, we show that the overexpression of thymic stromal lymphopoietin (TSLP) increased LTi cell numbers and restored LN development in IL-7(-/-) and RAG2(-/-) gamma(c)(-/-) mice. The TSLP-mediated LN restoration was strictly dependent on LTi cells and independent of lymphocyte colonization. Increased LTi cell numbers in the LN anlagen of RAG2(-/-) gamma(c)(-/-) TSLP transgenic mice were associated with the restoration of organizer cells, suggesting that LTi cell number is a critical parameter for LN organogenesis. Our results shed light on the minimal cellular requirement for LN development during ontogeny. We show that the presence of LTi and organizer cells, but not of peripheral lymphocytes, is critical for LN development and persistence and further suggest that the IL-7 signaling pathway regulates LN organogenesis by controlling the size of the LTi cell pool.