In addition to the primary culturing of cancer stem cells (CSCs) from tumor tissues, CSCs are found in established tumor cell lines. However, it is unclear how culture conditions affect CSC enrichment. Additionally, the differentiation potential of cell line-derived CSCs has not been well studied. In our study, the glioblastoma cell lines LN229, T98G, U251n and U87, were cultured as spheres in serum-containing medium (serum spheres) or serum-free medium (serum-free spheres). We found that LN229 and U251n cells expressed multiple stem cell markers such as Nestin, Sox2, Musashi-1 and CD44, and their serum spheres expressed even higher levels of Nestin, Sox2 and Musashi-1 compared to monolayer cells and serum‑free spheres. LN229 and U251n cells showed higher migration and colony formation potential compared to T98G and U87 cells, which did not express Nestin, Sox2 and Musashi-1. Serum spheres of LN229 and U251n cells also exhibited higher resistance to temozolomide compared to serum-free spheres. All tumor cell lines showed neuronal differentiation (Tuj-1 positive). Only U251n serum spheres exhibited both astrocytic (GFAP‑positive) and neuronal differentiation. We conclude that sphere culture in serum-containing medium provides the most efficient enrichment of cancer stem cells. U251n cells are distinguished from other tumor cells due to their potential for multilineage differentiation.