Few studies on the biologic and molecular properties of pediatric glioblastoma have been performed. Until now, differential genomic analysis of CD133+ve and CD133-ve fractions has not been described in pediatric glioma. We hypothesize not only that the presence of CD133 could be the source of tumor resistance but also that maintenance of this molecule by hypoxia dictates cellular and molecular behavior. From a series of human glioblastoma biopsies investigated, only one, IN699 (from a pediatric glioblastoma), generated greater than 4% of the total cell volume as CD133+ve cells. Using this pediatric glioblastoma, containing unprecedented high levels of the putative brain tumor stem cell marker CD133, as a study model, we report biologic and molecular characteristics of the parent culture and of CD133+ve and CD133-ve populations derived therefrom under atmospheric and hypoxic culture conditions. Immunocytochemistry and flow cytometry were performed with antigenic markers known to characterize neural stem cells and associated glioma behavior. Behavioral analysis was carried out using proliferation, adhesion, migration, and invasion assays. Cell cycle analysis and array comparative genomic hybridization were used to assess copy number profiles for parental cells and CD133+ve and CD133-ve fractions, respectively. With regard to invasion and proliferation, CD133+ve and CD133-ve fractions were inversely proportional, with a significant increase in invasive propensity within the CD133-ve cells (P < .005) and a significant increase in proliferation within CD133+ve cells (P < .005). Our observations indicate identical genomic imbalances between CD133+ve and CD133-ve fractions. Furthermore, our research documents a direct link between decreasing oxygen tension and CD133 expression.