Dendritic cells (DCs) are essential antigen-presenting cells (APCs) that consist of heterogeneous subsets, mainly classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). CD205, an endocytic type I C-type lectin-like molecule that belongs to the mannose receptor family, is mainly expressed on CD8α(+) cDCs. However, it is unclear how CD205(+) cDCs control immune responses in vivo. To evaluate the contribution of CD205(+) cDCs to the immune system, we engineered knock-in (KI) mice that express the diphtheria toxin receptor (DTR) under the control of the Cd205 gene, which allows the selective conditional ablation of CD205(+) cDCs in vivo. Conditional ablation of CD205(+) cDCs impaired the antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross presentation of soluble antigen. Upon microbial infection, CD205(+) cDCs contributed to the cross priming of CD8(+) T cells for generating antibacterial CTLs to efficiently eliminate pathogens. Here, we provide a protocol for the generation of bone marrow WT/CD205-DT chimeric mice, depletion of CD205(+) DCs and assessment of depletion efficiency, and protocols for in vivo cross presentation assay, CTL generation assay, and antibacterial immunity assay.