Recent studies revealed that the lymphotoxin/lymphotoxin beta receptor (LT)/LTbetaR system activates the noncanonical nuclear factor-kappaB (NF-kappaB) signaling pathway involving I kappa B kinase 1/I kappa B kinase alpha (IKK1/IKKalpha) and NF-kappaB-inducing kinase (NIK) to direct processing of the nfkappab2 protein p100 to yield RelB:p52 complexes. Despite the biochemical evidence, LT-, RelB-, p52-deficient mice show discrepant phenotypes. We now demonstrate that p105/p50 also constitutes an important pathway for LTbetaR signaling. Our studies revealed that mice deficient in either p50 or p52 have defects in the formation of inguinal lymph nodes (LNs), but that the complete defect in lymph node formation and splenic microarchitecture seen in LT-deficient mice is recapitulated only in mice deficient in both p50 and p52. Biochemically, we find not only that both p50- and p52-containing NF-kappaB activities are induced by LTbetaR signaling, but that the induction of NF-kappaB-containing complexes by LTbetaR engagement is perturbed in single knockouts. Importantly, the LTbetaR can additionally activate the less well-characterized p52:RelA and p50:RelB pathways, which play pivotal roles in vivo for the development and organization of lymphoid structures. Our genetic, cellular, and molecular evidence points toward a model of LT-mediated NF-kappaB regulation in which p105/p50 and p100/p52 have distinct and coordinating molecular specificities but differ in the upstream signaling pathways that regulate them.