Normal human embryonic stem cells (hESCs) can develop neoplastic cancer stem cell (CSC) properties after coculture with transformed hESCs in vitro. In the present study, the influence of the tumor microenvironment on malignant transformation of bone marrow stromal cells (BMSCs) was studied after allografting a mixture of enhanced green fluorescent protein (EGFP)-labeled BMSCs and C6 glioma cells into the rat brain to understand the influence of the cellular environment, especially the tumor environment, on the transformation of grafted BMSCs in the rat brain. We performed intracerebral transplantation in the rat brain using EGFP-labeled BMSCs coinjected with C6 tumor cells. After transplantation, the EGFP-labeled cells were isolated from the tumor using fluorescence-activated cell sorting, and the characteristics of the recovered cells were investigated. Glioma-specific biomarkers of the sorted cells and the biological characteristics of the tumors were analyzed. The BMSCs isolated from the cografts were transformed into glioma CSCs, as indicated by the marked expression of the glioma marker GFAP in glioma cells, and of Nestin and CD133 in neural stem cells and CSCs, as well as rapid cell growth, decreased level of the tumor suppressor gene p53, increased level of the oncogene murine double minute gene 2 (MDM2), and recapitulation of glioma tissues in the brain. These data suggest that BMSCs can be transformed into CSCs, which can be further directed toward glioma formation under certain conditions, supporting the notion that the tumor microenvironment is involved in transforming normal BMSCs into glial CSCs.