Aldehyde dehydrogenase 1 (ALDH1) has been used to isolate tumorigenic stem-like cells in a large number of tumors, including glioblastoma multiforme (GBM). We recently showed that human glioblastoma cells with high ALDH1 (ALDH1(high)) activity contain stem-cell-like characteristics. In the study reported here, we isolated established and primary human glioblastoma cells based on their ALDH1 expression. When tested for asymmetric division, only cells with ALDH1(high) expression were able to restore heterogeneous populations after a few days, whereas cells with ALDH1(low) levels could not. Most interestingly, the capacity of cells with ALDH1(low) levels to divide asymmetrically into cells with either ALDH1(high) or ALDH1(low) expression could be restored after exposure to hypoxic culture conditions. Consequently, we found neurosphere formation reinstated in posthypoxic, formerly ALDH1(low), cells. The direct involvement of ALDH1 could be confirmed by ALDH1 small hairpin ribonucleic acid (shRNA) knockdown, suggesting ALDH1 as an intracellular marker for the identification and isolation of stem-like glioblastoma cells. In summary, we show that ALDH1 expression correlates well with asymmetric division capacity and tumor sphere formation. Furthermore, we demonstrated that hypoxic culture conditions induce and/or upregulate ALDH1 expression in established and primary GBM cell lines.