In vitro cell culture is a routinely used method which is also applied for in vitro modeling of various neurological diseases. On the other hand, media used for cell culture are often not strictly standardized between laboratories, which hinders the comparison of the obtained results. Here, we compared the effects of homocysteine (Hcy), a molecule involved in neurodegeneration, on immature cells of the nervous system cultivated in basal medium or media supplemented by either fetal bovine serum or basic fibroblast growth factor. The number of cells in basal media supplemented with basic fibroblast growth factor (bFGF) was 2.5 times higher in comparison to the number of cells in basal media supplemented with fetal bovine serum (FBS). We also found that the neuron-specific β-3-tubulin protein expression dose dependently decreased with increasing Hcy exposure. Interestingly, bFGF exerts a protective effect on β-3-tubulin protein expression at a concentration of 1000 µM Hcy compared to FBS-treated neural stem cells on Day 7. Supplementation with bFGF increased SOX2 protein expression two-fold compared to FBS supplementation. GFAP protein expression increased five-fold on Day 3 in FBS-treated neural stem cells, whereas on Day 7, bFGF increased GFAP expression two-fold compared to FBS-treated neural stem cells. Here, we have clearly shown that the selection of culturing media significantly influences various cellular parameters, which, in turn, can lead to different conclusions in experiments based on in vitro models of pathological conditions.