Although CD133 has been proposed as a marker for brain tumor-initiating cells, studies show that a tumorigenic potential exists among CD133- glioma cells as well. However, it is not established whether the ability of CD133- cells to form tumors is a property confined to a small subpopulation, rather than a common trait associated with most glioma cell types. Thus, we used lentiviral vectors expressing green fluorescent protein under lineage-specific promoters to identify CD133- glioma cells expressing Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Flow cytometry analysis showed the presence of CD133- subpopulations expressing these markers in glioma cell lines and in primary cultures from human glioblastoma (GBM) biopsies. Moreover, analysis of cell cycle distribution showed that subgroups expressing Nestin, GFAP, and NSE uniformly contained actively cycling cells, when cultured in serum-containing medium and stem cell medium. These subpopulations were fluorescence-activated cell sorted from CD133- U373 glioma cells and implanted intracerebrally in severe combined immunodeficient mice. Moreover, we implanted Nestin-, GFAP-, and NSE-positive glioma cells sorted from a human GBM biopsy, following removal of CD133-positive cells. All the CD133- subpopulations produced tumors, with no significant differences in survival or tumor take rates. However, there was a trend toward lower take rates for CD133- glioma subpopulations expressing GFAP and NSE. These findings suggest that the ability to form tumors may be a general trait associated with different glioma cell phenotypes, rather than a property limited to an exclusive subpopulation of glioma stem cells.