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Although in vitro observations suggest that cross-presentation of antigens is mediated primarily by CD8α⁺ dendritic cells, in vivo analysis has been hampered by the lack of systems that selectively eliminate this cell lineage. We show that deletion of the transcription factor Batf3 ablated development of CD8α⁺ dendritic cells, allowing us to examine their role in immunity in vivo. Dendritic cells from Batf3^–/– mice were defective in cross-presentation, and Batf3^–/– mice lacked virus-specific CD8⁺ T cell responses to West Nile virus. Importantly, rejection of highly immunogenic syngeneic tumors was impaired in Batf3^–/– mice. These results suggest an important role for CD8α⁺ dendritic cells and cross-presentation in responses to viruses and in tumor rejection.