Lymph nodes provide specialized stromal microenvironments that support the recruitment and organization of T cells and B cells, enabling them to effectively participate in immune responses. While CD4(+)3(-) lymphoid tissue inducer cells (LTic's) are known to play a key role in influencing lymph node (LN) development, the mechanisms that regulate the development of stromal organizer cells are unclear. Here, we define an ontogenetic program of lymph node stromal cell maturation in relation to the requirement for LTic's. We also describe a lymph node reaggregation assay to study cell-cell interactions and lymphocyte recruitment to these organs that reproduces the in vivo events during lymph node development. In addition, analysis of the lymph node anlagen in normal and lymphotoxin a (LTa)-deficient embryos shows that LTa-mediated signaling is required to sustain proliferation and survival of stromal cells in vivo. Our data identify LTa-independent and LTa-dependent stages of lymph node development, and provide direct evidence for the role of LTic's during LN organogenesis.