Herpes simplex virus thymidine kinase (TK) gene-modified T cells are currently being evaluated in gene therapy clinical trials for the control of graft-versus-host disease (GVHD) after allogeneic BMT. Unfortunately, these trials have been limited by a consistent failure of the ex-vivo manipulated T cells to survive and function properly in vivo. We recently developed a technique for retrovirally transducing and selecting murine T cells with a novel chimeric CD34-TK fusion suicide gene that preserves their alloreactivity after allogeneic BMT. In this study, we assessed the trafficking, survival, and GVHD-inducing potential of ex vivo manipulated murine T-cells in fully allogeneic transplant recipients by in vivo bioluminescence imaging (BLI) with two novel reporter vectors. The first vector encodes a fusion protein comprised of click beetle red (CBR) luciferase and EGFP (CBR/EGFP). In the second vector, we inserted a click beetle green (CBG) luciferase between CD34 and TK in our chimeric suicide gene (CD34/CBG/TK). Murine T cells, stimulated 24 h with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads), were transduced with Phoenix-Eco-derived CBR/EGFP or CD34/CBG/TK retrovirus and purified to >85% using a MoFlo cell sorter or CD34 immunomagnetic selection 48 h post-infection. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted C57BL/6 (B6) bone marrow supplemented with either 1e6 CBR/EGFP or CD34/CBG/TK purified B6 T cells. The CBR/EGFP BLI signal was significantly increased over background at 24 h post-injection, with the allogeneic T cells localizing primarily to the spleen and secondary lymph nodes. Over the next 2–3 days the CBR/EGFP+ cells migrated to the entire intestinal area followed rapidly by infiltration of the skin. Overall, the CBR/EGFP BLI signal increased nearly 3 orders of magnitude between days 1 and 8 post-BMT, remained steady for a week, and then only gradually declined over the next month (only a 3-fold decrease between days 14 to 42 post-BMT). Consistent with GVHD, these mice lost >20% of their pretransplant body weight and exhibited impaired lymphoid reconstitution. We observed similar trafficking and GVHD-inducing potential when CD34/CBG/TK gene-modified T cells were injected into BALB/c recipients. However, the maximum BLI signal intensity from the CD34/CBG/TK T cells was decreased nearly 2 orders of magnitude compared to the CBR/EGFP-modified T cells. Nevertheless, we were still able to demonstrate a significant reduction in BLI signal intensity when recipients of CD34/CBG/TK-modified allogeneic T cells were treated with ganciclovir (GCV) from days 1 to 7 post-BMT. This observation is consistent with in vitro cell sensitivity assays, which demonstrated that cells modified with the CD34/CBG/TK reporter gene retain TK activity similar to CD34-TK modified cells. In summary, this study demonstrated by in vivo BLI that allogeneic murine T cells activated and expanded ex vivo with CD3/CD28 beads retain significant GVHD-inducing potential and can be eliminated by HSV-TK/GCV suicide gene therapy.