Lymphotoxin-α-deficient (LT-α^−/−) mice manifest congenital absence of lymph nodes (LNs) and Peyer’s patches and disturbed spleen follicle structure. The splenic white pulp areas show loss of discrete T and B lymphocyte zones, of follicular dendritic cell (FDC) clusters, and of germinal centers (GCs). Tumor necrosis factor receptor I-deficient (TNFR-I^−/−) mice show similar absence of FDC clusters and GCs but retain segregation of T and B cell zones. Rarely are mesenteric LNs found in LT-α^−/− mice. These mesenteric LNs show segregation of T and B cell zones similar to wild-type mice. In contrast, mesenteric LNs in TNFR-I^−/− mice manifest grossly disturbed organization of T and B cells. Both LT-α^−/− and TNFR-I^−/− mice lacked FDC clusters in LNs and spleen. Interestingly, although both LT-α^−/− and TNFR-I^−/− mice that had been immunized with sheep red blood cells failed to form GCs in the spleen, they both developed GC-like clusters of peanut agglutinin-positive (PNA⁺) cells in their LNs. Furthermore, when lethally irradiated recombination activating gene (RAG)-1-deficient (RAG-1^−/−) mice that had received spleen cells from LT-α^−/− mice were immunized with sheep red blood cells, they failed to generate PNA⁺ clusters in the reconstituted spleen but showed robust PNA⁺ clusters in the reconstituted LNs. These data demonstrate that the signals that regulate the development of distinct T and B cell zones as well as the signals that regulate B cell activation to produce clusters of PNA⁺ cells differ between the spleen and LNs.