Signaling through Notch receptors and their transcriptional effector RBP-J is essential for lymphocyte development and function, whereas its role in other immune cell types is unclear. We tested the function of the canonical Notch–RBP-J pathway in dendritic cell (DC) development and maintenance in vivo. Genetic inactivation of RBP-J in the bone marrow did not preclude DC lineage commitment but caused the reduction of splenic DC fraction. The inactivation of RBP-J in DCs using a novel DC-specific deleter strain caused selective loss of the splenic CD8⁻ DC subset and reduced the frequency of cytokine-secreting CD8⁻ DCs after challenge with Toll-like receptor ligands. In contrast, other splenic DC subsets and DCs in the lymph nodes and tissues were unaffected. The RBP-J–deficient splenic CD8⁻ DCs were depleted at the postprogenitor stage, exhibited increased apoptosis, and lost the expression of the Notch target gene Deltex1. In the spleen, CD8⁻ DCs were found adjacent to cells expressing the Notch ligand Delta-like 1 in the marginal zone (MZ). Thus, canonical Notch–RBP-J signaling controls the maintenance of CD8⁻ DCs in the splenic MZ, revealing an unexpected role of the Notch pathway in the innate immune system.