Incorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O⁶-benzylguanine (O⁶BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)–transduced autologous CD34⁺-selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O⁶BG and temozolomide resulted in a multilineage increase in gene-modified repopulating cells with marking levels of greater than 98% in granulocytes. MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in conjunction with O⁶BG and temozolomide in clinical trials.