Therapeutic failure in glioblastoma (GBM) is increasingly attributed not only to tumor cell-intrinsic factors but also to the adaptive/supportive tumor microenvironment that nurtures glioma stem cells (GSCs) and drive therapy resistance. GSCs reside within specialized niches shaped by extracellular matrix architecture, stromal interactions, metabolomic gradients, and immune-modulatory cues, enabling their survival, plasticity, and repopulation following conventional therapy. Effective targeting of GBM therefore requires strategies that disrupt both GSC-intrinsic niche and the supportive microenvironment context that limit drug penetration, retention, and therapeutic benefit. Traditional two-dimensional (2D) culture systems fail to capture these spatial and biological complexities, resulting in poor clinically actionable predictive power for successful outcomes. In contrast, three-dimensional (3D) models offer an opportunity to recapitulate relevance. Building on this context, this chapter highlights recent advances that integrate nanotechnology with stem cell-based 3D GBM organoid platforms to enable effective therapeutic delivery and resistance niche-level targeting. We discuss the design and functional evaluation of nanoparticle systems engineered for deep tumor penetration and selective delivery, including polymeric nanoparticles, mesoporous silica nanoparticles, and ultrasmall gold nanostructures. Emphasis is placed on mesenchymal and neural stem cell-mediated nanodelivery, biomimetic hydrogel- and nanofiber-based scaffolds for recreating GSC-associated niche, and advanced analytical readouts including electron microscopy, confocal Z-stack imaging, and ICP-MS. Collectively, this chapter presents a translational framework for leveraging 3D models and stem cell-directed nanotechnologies as preclinical tools to overcome therapy resistance and improve therapeutic outcomes in GBM.