Adoptive immunotherapy is a promising cancer treatment that entails infusion of immune cells manipulated to have antitumor specificity， in vitro. Antigen-specific cytotoxic T lymphocytes are the main executors of transformed cells during cancer immunotherapy. To induce antigen-specific cytotoxic T lymphocytes， we developed artificial antigen-presenting cells (aAPCs) by engineering K562 cells with electroporation to direct the stable expression of HLA-A∗0201， CD80， and 4-1BBL. Our findings demonstrate that after three stimulation cycles， the aAPCs promoted the induction of antigen-specific cytotoxic T lymphocytes with a less differentiated "young" phenotype， which enhanced immune responses with superior cytotoxicity. This novel， easy， and cost-effective approach to inducing antigen-specific cytotoxic T lymphocytes provides the possibility of improved cancer therapies.