In vivo assay to evaluate anti-cancer immunotherapy at the pre-clinical phase is eagerly needed. We currently established xenotransplantation-based method to analyze in vivo priming of cancer-antigen-specific human cytotoxic T lymphocytes (CTLs). We transplanted human peripheral T cells and analyzed priming of CTLs in NOG mice. Half of the mice engrafted with bulk lymphocytes including CD4 + T cells died before analysis probably due to xenoreactive graft versus host disease. All of the mice engrafted with purified CD8 + T cells survived until the analysis, and successful engraftment was observed in 80% of recipient mice. Thus, transfer of purified CD8 + T cells is sufficient and safer than that of bulk lymphocytes. To add antigenic stimulation to the CD8 + T cells in vivo, injection of antigenic peptide-loaded and monocyte-derived autologous dendritic cells (DCs) was simultaneously done and repeated 7 days later. The DC-based vaccinization resulted in efficient priming of HLA class I-restricted and MART1, WT1 or CMV peptides-specific CTLs in the recipient mice. This system may be useful to evaluate the stimulation of antigen-specific human CTLs in vivo.