Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.