To treat leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the possibility of immunotherapy using donor CD8+ T cells that were generated by stimulating leukemic cell-derived dendritic cells (leukemic-DCs) or leukemic cell lysate pulsed donor cell-derived DCs (donor-DCs). Leukemic- and donor-DCs were generated from mononuclear cells of patients and CD14+ cells of HLA-matched donors, respectively. The expression of CD80, CD83, CD86, CD1a, and CD40 on leukemic-DCs was significantly lower than that on donor-DCs. Donor-DCs exhibited a higher capacity to stimulate allogeneic T cells compared with leukemic-DCs. Donor CD8+ T cells stimulated by leukemic- or donor-DCs were more cytotoxic than unprimed CD8+ T cells, and slightly higher cytotoxicity was observed with donor-DCs compared to leukemic-DCs. This study indicates that leukemic- or donor-DCs pulsed with leukemic cell lysates can effectively prime donor cytotoxic T cells in vitro, and that they may be used as a potential alternative tool for treating leukemic patients who relapse after allogeneic HSCT.