Apoptotic cell clearance by dendritic cells (DCs) plays a crucial role in the maintenance of self-tolerance. In spleen, CD8α+ DCs are thought to be responsible for this phenomenon by phagocytosing circulating apoptotic cells. However, as CD8α+ DCs are believed to be predominantly localized in the T cell zone, it remains unclear how these DCs phagocytose blood-borne apoptotic cells accumulated in the marginal zone (MZ). In this study, we identified a subpopulation of CD8α+ DCs responsible for tolerance induction to cell-associated Ags. Among splenic CD8α+ DCs, the CD103+, CD207+ subset was preferentially localized in the MZ and dominantly phagocytosed blood-borne apoptotic cells. After phagocytosis of apoptotic cells, this DC subset migrated into the T cell zone for cross-presentation of cell-associated Ags. Stimulation of TLRs induced the disappearance of this DC subset. Consequently, CD8α+ DCs neither phagocytosed injected apoptotic cells nor presented cell-associated Ags in mice treated with TLR ligands. Transient ablation of this DC subset by cytochrome c injection resulted in a failure of tolerance induction to cell-associated Ags, indicating that this DC subset is essential for tolerance induction by apoptotic cell clearance.