The noncanonical NF-kappa B pathway regulates the development and function of multiple organs and cell lineages. We have generated mice harboring a novel mutation in Nfkb2 that prevents the processing of the inhibitory precursor, p100, into the active subunit, p52. Mutant mice express a complex phenotype with abnormalities in a variety of tissues, and with a spectrum that is more severe than in mice carrying a targeted deletion of Nfkb2. Signaling through the noncanonical pathway is ablated due to the absence of p52, resulting in disorganized splenic architecture and disrupted B cell development. The inhibitory precursor form of NF-kappa B2 interacts with RelA, preventing activation of RelA dimers in response to both canonical and noncanonical stimuli, which in combination with p52 deficiency, results in defective lymph node formation and bone homeostasis. These findings demonstrate a key role for NF-kappa B2 in the regulation of RelA activation and suggest overlap in the function of NF-kappa B members in canonical and noncanonical pathway signaling.