Induction of cytotoxic T lymphocytes (CTLs) for treatment of relapsed leukemia after allogeneic stem-cell transplantation is hindered by the laborious and time-consuming procedure of generating dendritic cells for antigen presentation. Artificial antigen-presenting cells (aAPCs) offer the advantage of being readily available in sufficient numbers, thus allowing for a highly standardized in vitro induction of CTLs. We generated aAPCs coated with anti-CD28 antibody (Ab) and either high-density (HD) or low-density (LD) major histocompatibility complex (MHC) class I molecules loaded with HA-1^H, a nonapeptide derived from the hematopoiesis-restricted minor histocompatibility antigen HA-1. HD- and LD-aAPCs potently induced HA-1^H–specific CD8⁺ CTLs from untouched CD8⁺ T cells of healthy donors. CTLs were subsequently purified by magnetic-activated cell sorting. HD- as well as LD-aAPC–induced CTLs exerted high HA-1^H–specific cytotoxicity, resembled T_c1 effector memory cells, survived a long time in vitro, and were expanded by a factor varying between 8.2 × 10⁴ and 51 × 10⁴. The T-cell receptor (TCR) repertoire of HA-1^H tetramer–positive CTLs was oligoclonal with a prominent usage of Vβ6. The TCR repertoire of tetramer-positive CTLs was distinct from and more restricted than that of tetramer-negative cells. These findings indicate that aAPCs are attractive tools for the ex vivo generation of HA-1^H–specific CTLs suitable for immunotherapy of relapsed leukemia.