NF-kappaB-inducing kinase (NIK) is responsible for activation of the non-canonical p100 processing pathway of NF-kappaB activation. This kinase has been shown to be critical for activation of this pathway after signaling through several TNF family members including CD40. The functional importance of this pathway in CD40 and TLR-induced dendritic cell (DC) differentiation was studied in vivo in the alymphoplasia (Aly) mouse. The Aly mouse expresses a mutant NIK molecule that prohibits the induction of the non-canonical pathway. We show that while MHC class II presentation and in vivo migration of Aly DCs is intact, these cells are unable to cross-prime CD8+ T cells to exogenous Ag. Gene expression array analysis of DCs matured in vivo indicates multiple defects in Ag processing pathways after maturation and provide a global view of the genes that are regulated by the NF-kappaB2 pathway in DCs. These experiments indicate a possible role for NIK in mediating cross-priming of soluble Ag. In addition, our findings explain the profound immune unresponsiveness of the Aly mouse.