Objective: Glioblastoma is a malignant and aggressive brain tumor characterized by deregulation of certain transcription factors. One of its main traits, cellular heterogeneity, probably originates from tumor stem cells, a treatment-resistant and poorly differentiated cell population capable of continuous proliferation and formation of phenotypically distinct cells. The exceptional adaptive abilities and drug resistance of glioblastoma result from peculiarities of gene expression in tumor cells. Studying the expression of key transcription factors and mechanisms of tumor stem cell differentiation can help identify molecular pathways important for glioblastoma therapy. Methods: Changes in expression of transcription factors MYCN, MYCC, PHOX2A, and PHOX2B, which are involved in cell cycle regulation and differentiation, were evaluated using real-time PCR in response to changes in cultivation conditions. Results and Discussion: Primary cultures grown in medium with fetal bovine serum acquired morphology and gene expression similar to cell lines. Moreover, all cell lines and primary cultures of glioblastoma under standard conditions differed in expression profiles of the studied genes. Primary cultures with the proneural subtype demonstrated higher PHOX2A expression than tumors with the mesenchymal subtype. Often, PHOX2B expression was not detected. Interestingly, in response to changes in cultivation conditions, all lines and cultures demonstrated multiple increases in MYCN expression, as well as opposite responses of PHOX2A and PHOX2B. Conclusions: Under the new conditions, there is a shift away from the initial molecular parameters of the tumor toward less accurate in vitro models of glioblastoma—cell lines. Moreover, changes in MYCN, PHOX2A, and PHOX2B expression in response to altered cultivation conditions indicate a possible role of these genes in glioblastoma stress resistance, making them important targets for further research to identify therapeutic targets for glioblastoma.