<p>Glioblastoma remains one of the most lethal malignancies, largely due to its resistance to standard chemotherapy such as temozolomide. This study investigates a novel resistance mechanism involving glioblastoma stem cells (GSCs) and the polarization of M2-type macrophages, mediated by the extracellular vesicle (EV)-based transfer of Clusterin. Using 6-week-old male CD34⁺ humanized huHSC-(M-NSG) mice (NM-NSG-017) and glioblastoma cell lines (T98G and U251), we demonstrated that GSC-derived EVs enriched with Clusterin induce M2 macrophage polarization, thereby enhancing temozolomide resistance in glioblastoma cells. Single-cell and transcriptome sequencing revealed close interactions between GSCs and M2 macrophages, highlighting Clusterin as a key mediator. Our findings indicate that Clusterin-rich EVs from GSCs drive glioblastoma cell proliferation and resistance to temozolomide by modulating macrophage phenotypes. Targeting this pathway could potentially reverse resistance mechanisms, offering a promising therapeutic approach for glioblastoma. This study not only sheds light on a critical pathway underpinning glioblastoma resistance but also lays the groundwork for developing therapies targeting the tumor microenvironment. Our results suggest a paradigm shift in understanding glioblastoma resistance, emphasizing the therapeutic potential of disrupting EV-mediated communication in the tumor microenvironment.</p>