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The rat 9L gliosarcoma is a widely used syngeneic rat brain tumor model that closely simulates glioblastoma multiforme when implanted in vivo. In this study, we sought to isolate and characterize a subgroup of cancer stem‐like cells (CSLCs) from the 9L gliosarcoma cell line, which may represent the tumor‐initiating subpopulation of cells. We demonstrate that these CSLCs form clonal‐derived spheres in media devoid of serum supplemented with the mitogens epidermal growth factor and basic fibroblast growth factor, express the NSC markers Nestin and Sox2, self‐renew, and differentiate into neuron‐like and glial cells in vitro. More importantly, these cells can propagate and recapitulate tumors when implanted into the brain of syngeneic Fisher rats, and they display a more aggressive course compared with 9L gliosarcoma cells grown in monolayer cultures devoid of mitogens. Furthermore, we compare the chemosensitivity and proliferation rate of 9L gliosarcoma cells grown as a monolayer to those of cells grown as floating spheres and show that the sphere‐generated cells have a lower proliferation rate, are more chemoresistant, and express several antiapoptosis and drug‐related genes, which may prove to have important clinical implications.  Disclosure of potential conflicts of interest is found at the end of this article.

Spheres Isolated from 9L Gliosarcoma Rat Cell Line Possess Chemoresistant and Aggressive Cancer Stem‐Like Cells