Although the concept of cancer stem cells or cancer-initiating cells had created a new paradigm for the treatment of malignant tumors, it remains unclear how cancer-initiating cells can be eradicated. We have previously reported that the transforming growth factor-β (TGF-β)-Sox4-Sox2 pathway is essential for glioma-initiating cells to retain their stemness, and inhibition of TGF-β signaling may lead to differentiation of glioma-initiating cells (Ikushima, H., Todo, T., Ino, Y., Takahashi, M., Miyazawa, K., and Miyazono, K. (2009) Cell Stem Cell 5, 504-514). Here we demonstrate that Oct4 plays essential roles in retention of the stemness properties of glioma-initiating cells through positive regulation of Sox2 expression. We also show that, in glioma-initiating cells, Oct4 is associated with Sox4 and that Oct4-Sox4 complexes cooperatively activate the enhancer activity of the SOX2 gene. In contrast, in fetal neural progenitor cells, Sox2 expression is enhanced by transcriptional complex containing Sox2 protein itself, and this self-reinforcing loop of Sox2 appears to be disrupted in glioma-initiating cells, suggesting that Sox2 expression in glioma-initiating cells is differently regulated from that in neural progenitor cells. Our findings reveal differences between glioma-initiating cells and fetal neural progenitor cells and may open the way to depriving glioma-initiating cells of tumorigenic activity without affecting normal tissues.