RelB, a member of the NF-κB/Rel family of transcription factors, has been implicated in the constitutive expression of KB-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive κB-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. ReIB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, ReIB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-κB/Rel family.