More efficient isolation and identification of cancer stem cells (CSCs) would help in determining their fundamental roles in tumor biology. The classical tool for this purpose is anchorage-independent tumorsphere culture. We compared the effects of differently textured culture plates and serum deprivation on the acquisition of CSC properties of A172 glioblastoma cells. Cells were cultured on standard polystyrene-treated plates, ultra-low attachment, poly (2-hydroxyethyl methacrylate)-coated plates, and 1% agar-coated plates with 10% serum or in serum-free glioblastoma sphere medium (GBM). Based on mitochondrial reductase activity and subG1 proportions, non-adherent conditions had a greater impact on A172 cell viability than serum deprivation. Among the stemness-related genes, SOX-2 expression was significantly upregulated by serum deprivation under non-adherent conditions, while several epithelial-to-mesenchymal transition (EMT)-related genes were less dependent on serum. In addition, reactive oxygen species (ROS) accumulation in A172 cells was significantly increased in GBM under non-adherent conditions. Despite the correlation between SOX-2 induction and ROS accumulation, treatment with the ROS scavenger N-acetyl-l-cysteine did not prevent SOX-2 expression, suggesting that ROS accumulation is not an essential requirement for induction of SOX-2. Our results suggested that cultivation of cancer cells under conditions of serum deprivation in an anchorage-independent manner may enrich SOX-2-expressing CSC-like cells in vitro.