Cross-presentation as a fundamental pathway of activating CD8⁺ T cells has been well established. So far the application of this concept in vivo is limited, and the mechanisms that specialize CD8⁺ dendritic cells (DCs) for this task are not fully understood. Here we take advantage of the specific cytosolic export feature of cross-presenting DCs together with the property of cytosolic cytochrome c (cyt c) in initiating Apaf-1-dependent apoptosis selectively in cross-presenting DCs. A single i.v. injection of cyt c in B6 mice produced a 2- to 3-fold reduction in splenic CD8⁺ DCs but not in Apaf-1-deficient mice. Functional studies both in vivo and in vitro showed that cyt c profoundly abrogated OVA-specific CD8⁺ T cell proliferation through its apoptosis-inducing effect on cross-presenting DCs. More importantly, in vivo injection of cyt c abolished the induction of cytotoxic T lymphocytes to exogenous antigen and reduced subsequent immunity to tumor challenge. In addition, only a proportion of CD8⁺ DCs that express abundant IL-12 and Toll-like receptor 3 were efficient cross-presenters. Our data support the hypothesis that cross-presentation in vivo requires cytosolic diversion of endocytosed proteins, conferring cross-presentation specialization to a proportion of CD8⁺ DCs. We propose that DCs incapable of such transfer, even within the CD8⁺ DC subset, are unable to cross-present. Our model opens an avenue to specifically target cross-presenting DCs in vivo for manipulating cytotoxic T lymphocyte responses toward infections, tumors, and transplants.