Tumor hypoxia has been attributed to play a crucial role in tumorigenesis and therapeutic resistance. Recently, it has been suggested that hypoxia leads to and maintains the undifferentiated state of tumor stem cells, thereby contributing to chemoresistance. The aim of the present study is to investigate the influence of hypoxia on the protein expression of a panel of stem cell and chemoresistance markers using in vivo-like multicellular tumor spheroids derived from a glioblastoma short-term culture with tumor stem cell properties (SJ-1) as well as a conventional glioblastoma cell line (U87). Spheroids were formed in 21% and 1% O(2) in serum-free medium. The immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67), and stem cell markers (CD133, podoplanin, Bmi-1, nestin, Sox-2) as well as markers related to chemoresistance (MGMT, TIMP-1, Lamp-1, MRP1, MDR-1). As spheroids derived in hypoxia were smaller than in normoxia, a set of experiments was included in which the culturing time of hypoxic spheroids was extended to obtain equally sized spheroids. The results showed that expression of HIF-1α and HIF-2α was increased in hypoxia, whereas Ki-67 was reduced. Expression of stem cell markers CD133, podoplanin, Bmi-1, and nestin was increased in hypoxia, whereas Sox-2 was increased in SJ-1 only. TIMP-1 and Lamp-1 were increased in both SJ-1 and U87. In conclusion, the tumor cell phenotype related to stemness, and thereby potentially to chemoresistance, seems to depend on the oxygen tension, suggesting that development of therapeutic strategies targeting tumor stem cells should take oxygen tension into account.