Glioblastoma (GBM) is the most prevalent and lethal primary intrinsic brain cancer. The disease is essentially incurable, with glioblastomas characterized by resistance to both chemotherapy and radiotherapy, as well as by rapid tumor progression, all of which are mainly ascribed to glioma stem-like cells (GSLCs). In the present study, an improved model that is more similar to clinical GBM was constructed. Twenty clinical glioma samples were collected to obtain primary low-grade tumor cells. The cells were either maintained in serum-free medium as primary glioma-based cells (PGBCs) or cultured in the same medium with CHIR99021 as GSLCs. Then, the molecular and ultrastructural differences between the two cell groups were determined. Furthermore, the proliferation and migration of the GSLCs were examined and the potential mechanisms were investigated. Finally, temozolomide resistance in vitro and in the mouse model was assessed to study the properties of the induced GSLCs. The primary low-grade tumor cells extracted from surgical samples were enriched with GSLC properties, with high expression levels of CD133 and Nestin in 100 nM CHIR99021. The GSLCs exhibited high proliferation and migration. Furthermore, the expression of the PI3K/AKT signaling pathway and that of related genes and proteins were significantly enhanced by CHIR99021. The animal study also revealed high levels of STAT3, mTOR, NF-kappa B, and VEGF in the GSLC-transplanted mice. CHIR99021 could stably enhance GSLC properties in patient-derived glioma samples. It may provide a useful model for further study, helping to understand the pathogenesis of therapeutic resistance and to screen drug candidates.