Retroviral transfer of the Herpes Simplex thymidine kinase (HSVTK) suicide gene to donor T cells has been used as a safety strategy against graft-versus-host disease following allogeneic stem cell transplantation. The feasibility of this strategy in human studies has been demonstrated, but a number of limitations have become apparent. Preactivation of donor lymphocytes using mitogens or monoclonal antibodies is essential for retroviral transduction, but can compromise subsequent T-cell function in vivo. We report the application of lentiviral vectors for transduction of T cells in cytokine culture, without activation through the T-cell receptor. Using vectors encoding either enhanced green fluorescent protein or a truncated CD34/mutant HSVTK fusion selection/suicide construct, we investigated the properties of T cells after gene modification. We found that following cytokine stimulation, a fraction of T cells undergoes division, and transgene expression occurred predominantly in these cells. Antiviral and alloreactive responses were preserved in these populations, and in contrast to fully activated T cells, there was minimal perturbation of regulatory T-cell numbers. We conclude that the use of interleukin-7 for lentiviral transduction offers the greatest potential for gene transfer to T cells without loss of function, and is favored for the clinical production of suicide gene modified T cells.