Reticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-kappaB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type-specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-kappaB1 and C-Rel, Rel/NF-kappaB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1(-/-)C-Rel(-/-) cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1(-/-)C-Rel(-/-) pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9-mediated death of Nfkb1(-/-)C-Rel(-/-) pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-x(L)) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-kappaB transcription factors independently regulate TLR-9-mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-kappaB1 and c-Rel, while largely dispensable for TLR-9-induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.