Abstract High levels of the Rel/NF-κB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer’s patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB−/−) mice, in contrast to nfkb1−/−, but similar to nfkb2−/− mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB−/− splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB−/− spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.