Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8⁺ cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen–immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig–based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen–major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig–based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.