It has been suggested that stress stimuli from the microenvironment maintain a subset of tumor cells with stem-like properties, including drug resistance. Here, we investigate whether Sp1, a stress responsive factor, regulates sternness gene expression and if its inhibition sensitizes cancer cells to chemotherapy. Hydrogen peroxide- and serum deprivation-induced stresses were performed in glioblastoma (GBM) cells and patient-derived cells, and the effect of the Spl inhibitor mithramycin A (MA) on these stress-induced stem cells and temozolomide (TMZ)-resistant cells was evaluated. Sp1 and stemness genes were not commonly overexpressed in clinical GBM samples. However, their expression was highly induced by stress stimuli. Using MA, we demonstrated Spl as a critical sternness-related transcriptional factor protecting GBM cells against stress- and TMZ-induced death. Thus, Spl inhibition may prevent recurrence of malignant cells persisting after primary therapy. (C) 2017 Elsevier Inc. All rights reserved.