We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTβ-R and TNF-R signaling. Lymph nodes developed in LTα^−/− mice treated in utero with agonist anti-LTβ-R monoclonal antibody. Thus, LTβ-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTαβ/LTβ-R interaction were generated. Normal mice treated in utero with LTβ-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTα^−/− mice had disrupted cellular organization. Therefore, LTβ-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.