Tumor-specific cytotoxic T lymphocytes (CTLs) from patients with early-stage tumors are usually more efficient at attacking tumor cells than CTLs from the progressing tumor stages. The authors investigated the antitumor activity of CTLs from gastric cancer patents and healthy donors. In this study, peripheral blood lymphocytes (PBLs) from gastric cancer patients and healthy donors were stimulated with HLA-A matched allogeneic gastric cancer cells such as KATO-3, MKN45, and SGC7901. Three different populations of lymphocyte, p5-CTL-KATO, h4-CTL-MKN45, and h4-CTL-KATO, were induced and expanded. Flow cytometry analyses showed that 85.2% to 97.8% of these cells were CD3-positive and 45.5% to 51.2% were CD8-positive. The induced CTLs efficiently kill HLA-A2 or HLA-A24 gastric cancer cells through CTL-mediated cytotoxicity. However, no effects were observed for other cancer cells or HLA-A2 negative gastric cancer cells. The specific cytotoxicity of the induced CTLs was further confirmed by cold-target inhibition and monoclonal antibody blockage. These results suggest that CTL-mediated cytotoxicity specific for tumor cells can be produced by stimulating PBLs from healthy donors using HLA-A matched tumor cells, which will lead to the development of new immunotherapeutic strategies to kill cancer cells.