The DNA repair enzyme O6-methylguanine methyltransferase (MGMT) is a major determinant of glioma resistance to alkylating agents. Several strategies have been used to induce sensitization to alkylator-based treatments, including the direct MGMT inhibitor O6-benzylguanine (BG). However, replenishment of MGMT is often observed after the withdrawal of combined schedules of temozolomide (TMZ) and BG, thus preventing further treatment efficacy. In this study we investigated the potential mechanisms of resistance to combination treatment with TMZ and BG in the MGMT-proficient, p53-mutated (mt p53) T98G glioblastoma (GBM) cell line, looking for an effect on nuclear factor kappa B (NFκB) and mt p53, which are both transcriptional regulators of MGMT. The administration of TMZ alone led to minimal inhibition of T98G cell viability which was, however, enhanced with the addition of BG. This effect coincided with reduced expression of MGMT protein and transcript levels, and a decrease in cellular amount of NFκB and mt p53. However, withdrawal of the drugs led to an increase in cell viability, which was in parallel with repletion of MGMT protein and transcript levels and was also accompanied by elevated protein levels of NFκB and mt p53. Overall, these results suggest that NFκB and mt p53 induction may be responsible for the failure of BG to induce prolonged inhibition of direct repair in TMZ co-treated GBM cells with mt p53 status.