Mature dendritic cells (DCs) are potent antigen presenting cells (APCs) that have been used in vaccine studies and adoptive immunotherapy protocols. For many clinical studies DCs are derived from monocytes in the presence of cytokines, which are expensive and often unavailable for clinical use. Here we describe a cytokine independent method for the differentiation of monocytes into APCs for the reactivation of antigen-specific memory T cells from both healthy donors and cancer patients. Contact activation of monocytes resulted in secretion of proinflammatory cytokines, such as IL-8, and increased cell surface expression of costimulatory molecules. To determine if activated monocytes (actMo) like DC can reactivate antigen-specific CTL, they were transduced with adenoviral vectors encoding the subdominant Epstein Barr virus antigens, latent membrane proteins (LMP) 1 and 2, which are expressed in Epstein Barr virus-positive malignancies. Stimulation of peripheral blood mononuclear cells with LMP1- and LMP2-expressing actMo activated LMP1- and LMP2-specific T cells, which could be further expanded with LMP1 or LMP2 expressing lymphoblastoid cell lines. The use of actMo as APCs simplifies the production/manufacture of antigen-specific T cells for clinical trials.