To characterize the replicative capacity of human central memory (T CM) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4+ T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR* 0401 CD4 T CM were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T CM to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T CM cells, yielding cultures with> 80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to> 99% Ag-specific T cells. After in vitro culture for 3–12 wk, the flu-specific CD4 T CM had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T CM were capable of 20–40 mean population doublings in vitro, and the expanded cells produced IFN-γ, IL-2, and TNF-α in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T CM that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.