A subset of quiescent tumor cells, labeled by the Nes-ΔTK-GFP transgene, is responsible for glioblastoma (GBM) recurrence after chemotherapy. This study used a genetically engineered mouse model to show that after treatment with temozolomide (TMZ), which temporarily stops tumor growth, the Nes-ΔTK-GFP-positive cells give rise to new tumor cells. These cells are also found in GBM tumors and are resistant to TMZ. Ablation of these cells with ganciclovir significantly reduced tumor growth, and combined TMZ-ganciclovir treatment impeded tumor development. The data suggest that a relatively quiescent subset of endogenous glioma cells sustains long-term tumor growth by producing transiently proliferative cells. The Nes-ΔTK-GFP transgene specifically labels quiescent stem cells in the subventricular zone (SVZ) and can be used to ablate dividing neural progenitors with ganciclovir. When introduced into glioma-prone mice, the transgene marked a subset of tumor cells that were quiescent and resistant to TMZ. After TMZ treatment, these cells reinitiated proliferation, leading to tumor recurrence. Pulse-chase experiments using BrdU analogs showed that the majority of cells incorporating these analogs were GFP-positive, indicating that the Nes-ΔTK-GFP-positive cells were the source of new tumor cells. Chronic ganciclovir treatment effectively blocked neurogenesis by ablating quiescent cells as they entered the cell cycle. This treatment significantly prolonged survival and reduced tumor progression in glioma-prone mice. The study also showed that the Nes-ΔTK-GFP transgene did not affect tumor development or survival when introduced into the Mut7 genetic background. However, when combined with TMZ, the treatment significantly reduced tumor growth and improved survival. The study also examined the bystander effect of ganciclovir, where HSV-TK-expressing cells can induce death of neighboring non-TK-expressing cells. However, no significant bystander effect was observed in the tumor cells. The study concluded that the Nes-ΔTK-GFP-positive cells are the primary source of tumor growth and that their elimination through ganciclovir treatment effectively disrupts the continued production of tumor cells. The findings support the hypothesis that a hierarchical cell population, including quiescent stem cells, is responsible for glioblastoma recurrence after therapy.A subset of quiescent tumor cells, labeled by the Nes-ΔTK-GFP transgene, is responsible for glioblastoma (GBM) recurrence after chemotherapy. This study used a genetically engineered mouse model to show that after treatment with temozolomide (TMZ), which temporarily stops tumor growth, the Nes-ΔTK-GFP-positive cells give rise to new tumor cells. These cells are also found in GBM tumors and are resistant to TMZ. Ablation of these cells with ganciclovir significantly reduced tumor growth, and combined TMZ-ganciclovir treatment impeded tumor development. The data suggest that a relatively quiescent subset of endogenous glioma cells sustains long-term tumor growth by producing transiently proliferative cells. The Nes-ΔTK-GFP transgene specifically labels quiescent stem cells in the subventricular zone (SVZ) and can be used to ablate dividing neural progenitors with ganciclovir. When introduced into glioma-prone mice, the transgene marked a subset of tumor cells that were quiescent and resistant to TMZ. After TMZ treatment, these cells reinitiated proliferation, leading to tumor recurrence. Pulse-chase experiments using BrdU analogs showed that the majority of cells incorporating these analogs were GFP-positive, indicating that the Nes-ΔTK-GFP-positive cells were the source of new tumor cells. Chronic ganciclovir treatment effectively blocked neurogenesis by ablating quiescent cells as they entered the cell cycle. This treatment significantly prolonged survival and reduced tumor progression in glioma-prone mice. The study also showed that the Nes-ΔTK-GFP transgene did not affect tumor development or survival when introduced into the Mut7 genetic background. However, when combined with TMZ, the treatment significantly reduced tumor growth and improved survival. The study also examined the bystander effect of ganciclovir, where HSV-TK-expressing cells can induce death of neighboring non-TK-expressing cells. However, no significant bystander effect was observed in the tumor cells. The study concluded that the Nes-ΔTK-GFP-positive cells are the primary source of tumor growth and that their elimination through ganciclovir treatment effectively disrupts the continued production of tumor cells. The findings support the hypothesis that a hierarchical cell population, including quiescent stem cells, is responsible for glioblastoma recurrence after therapy.