The role of NF-κB-inducing kinase (NIK) in cytokine signaling remains controversial. To identify the physiologic functions of NIK, we disrupted the NIK locus by gene targeting. Although NIK-/- mice displayed abnormalities in both lymphoid tissue development and antibody responses, NIK-/- cells manifested normal NF-κB DNA binding activity when treated with a variety of cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and lymphotoxin-β (LTβ). However, NIK was selectively required for gene transcription induced through ligation of LTβ receptor but not TNF receptors. These results reveal that NIK regulates the transcriptional activity of NF-κB in a receptor-restricted manner.