Background: Malignant gliomas (MGs) remain largely incurable with a poor prognosis and an average survival of approximately 1 year. They are resistant to radiotherapy (RT) and temozolomide (TMZ). Clearly more effective treatments for MGs are needed. Oncolytic viruses are promising new agents used against experimental models of MGs and are now in several clinical trials. Myxoma virus is an excellent oncolytic virus because it is safe, selectively infecting and killing only cancer cells. We have shown that myxoma virus is efficacious in MGs and brain tumor initiating cells (BTICs) in vitro and in vivo, but the effect on survival in BTICs is not striking and only seen when combined with rapamycin. We think that manipulating myxoma virus will improve its therapeutic efficacy in MG, and hypothesize that genetic deletion of the anti-apoptotic gene M11L in Myxoma virus will enhance cell killing in MGs/BTICs and may be synergistic with TMZ and RT. Methods: We created the myxoma M11L knockout virus (M11L-KO) and tested its ability to kill MG cells and BTICs isolated from fresh human malignant gliomas. We have also examined the efficacy of M11L-KO virus on killing of BTICs when combined with TMZ and RT. Results: We found that M11L-KO is superior to WT-myxoma, produces a productive infection, and kills glioma cells and BTICs via apoptosis as shown by cleavage of PARP and caspase-3. As expected, the M11L protein in the WT virus does associate with the mitochondria, which is consistent with its role in inhibiting apoptosis. We have also found an additive effect of TMZ and RT with the M11L-KO virus compared to WT-myxoma. Finally, a microarray screen in BTICs showed several promising candidates in the intrinsic and extrinsic apoptosis pathways that are down- or up-regulated by M11L in WT-myxoma. Interestingly, our microarray data showed that TNFSF10 is up-regulated in BTICs infected with M11L-KO myxoma compared to cells infected with WT-myxoma, indicating its possible role in Myxv-M11L-KO-mediated killing of BTICs. Conclusions: These results will have clinical significance in the development of an experimental treatment in MGs patients.