Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. The GBM-specific tumor microenvironment (TME) plays a crucial role in tumor progression, immune escape, local invasion, and metastasis of GBM. Here, we demonstrate that hypoxia, reactive oxygen species (ROS), and differential concentration of glucose influence the expression of cytokines and chemokines, such as IL-6, IL-8, and IP-10, in human glial cell lines. Treatment with cobalt chloride (CoCl2) and hydrogen peroxide (H2O2) significantly increased the expression levels of IL-6, IL-8, and IP-10 in a dose-dependent manner in CRT-MG and U251-MG astroglioma cells, but not in microglia cells. However, we found strikingly different patterns of expression of cytokines and chemokines between H2O2-treated CRT-MG cells cultured in low- and high-glucose medium. These results suggest that astroglioma and microglia cells exhibit distinct patterns of cytokine and chemokine expression in response to CoCl2 and H2O2 treatment, and different concentrations of glucose influence this expression under either hypoxic or oxidant-enriched conditions.