O6-Benzylguanine (BG) potentiates temozolomide (TMZ) cytotoxicity in tumors by inactivating O6-alkylguanine DNA alkyltransferase but also increases toxicity in hematopoietic cells. To improve the hematopoietic cell tolerance to alkylating agents, we retrovirally transduced the BG-resistant mutant G156A methylguanine DNA methyltransferase gene (deltaMGMT) into hematopoietic progenitors and evaluated whether deltaMGMT expression in hematopoietic colony-forming units would result in greater drug resistance to TMZ. DeltaMGMT expression in human and mouse colony-forming units followed by BG treatment resulted in a >7.7-fold increase in the TMZ 90% inhibitory concentration (IC90) and a 5.6-fold increase in the 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) IC90 relative to untransduced cells. This degree of protection enabled deltaMGMT-transduced CD34 cells to become much more resistant to BG and TMZ than SW480 cells, which express high O6-alkylguanine DNA alkyltransferase and are normally resistant to TMZ or BCNU alone. DeltaMGMT-transduced long-term culture initiating cells were also resistant to the BG and TMZ combination, as were untransduced long-term culture initiating cells, suggesting that noncycling early progenitors may be partially protected from TMZ. These data indicate that retroviral transduction of deltaMGMT into hematopoietic progenitors followed by BG and TMZ treatment may selectively protect hematopoietic cells more efficiently than BCNU, allowing dose-intensive and repetitive therapy without the risk of cumulative myelosuppression.