The adoptive transfer of donor T cells that have been genetically modified to recognize leukemia could prevent or treat leukemia relapse after allogeneic HSCT (allo-HSCT). However, adoptive therapy after allo-HSCT should be performed with T cells that have a defined endogenous TCR specificity to avoid GVHD. Ideally, T cells selected for genetic modification would also have the capacity to persist in vivo to ensure leukemia eradication. Here, we provide a strategy for deriving virus-specific T cells from CD45RA(-)CD62L(+)CD8(+) central memory T (T(CM)) cells purified from donor blood with clinical grade reagents, and redirect their specificity to the B-cell lineage marker CD19 through lentiviral transfer of a gene encoding a CD19-chimeric Ag receptor (CAR). Virus-specific T(CM) were selectively transduced by exposure to the CD19 CAR lentivirus after peptide stimulation, and bi-specific cells were subsequently enriched to high purity using MHC streptamers. Activation of bi-specific T cells through the CAR or the virus-specific TCR elicited phosphorylation of downstream signaling molecules with similar kinetics, and induced comparable cytokine secretion, proliferation, and lytic activity. These studies identify a strategy for tumor-specific therapy with CAR-modified T cells after allo-HSCT, and for comparative studies of CAR and TCR signaling.