Glioblastomas are a heterogeneous group of tumors of neuroectodermal origin. Tumor cell cultures obtained from patients with glioblastomas differ in morphology and phenotype, as well as genetic characteristics. In the current work we used glioblastoma cell lines A172 and T98G (known since the 1970s) and the new glioblastoma lines R1 and T2 (obtained in 2010 from surgical material of patients). The purpose of this study was to evaluate the effect of the alkylating antitumor drug fotemustine (FM) after single and repeated exposure on the survival and proliferation of these glioblastoma cells, as well as the effect of FM on gene expression of growth factors, extracellular matrix proteins, mesenchymal cell markers, and transcription factor Zeb1. The effect of FM on A172 and R1 glioblastoma cells differs from its effect on T98G and T2 cells. LD50 was in an amount of 60 μg/mL for A172 cells and 250 μg/mL for T98G cells. Single exposure of A172 and R1 cell lines to 100 μg/mL of FM resulted in the death of all cells in culture. A172 and R1 cell exposure to sublethal doses of FM caused decreased activity of all studied genes. After incubation of T98G and T2 glioblastoma cells with FM in sublethal doses we identified two cell populations: proliferating cells and nonproliferating growth-arrested cells. Cell exposure to FM in doses over 300 μg/mL led to the predominance of growth-arrested cells in populations. These cells remained viable for 1.5 months. The effect of FM on T98G and T2 cell lines was accompanied by increased activity of all the studied genes. In conclusion, a population of growth-arrested, drug-resistant cells has been revealed in T98G and T2 cell cultures after exposure to FM. These cells exhibit high expression of genes encoding factors associated with the epithelial–mesenchymal transition. We discuss the possible impact of nonproliferating cells actively producing growth factors on the tumor microenvironment.