The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8⁺ T cells. The starting repertoire of CD8⁺ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8⁺ T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.