Recent studies have shown that a small subpopulation of stem-like cancer cells within most solid tumors are responsible for the malignancy of aggressive cancer cells, including tumorigenicity and relapse of solid tumors. These tumor cells may be enriched and maintained in vitro in the presence of growth factors (GFs), including epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), without serum in almost all solid tumor cell lines. In the present study, however, we found that the enrichment of cancer stem-like cells (CSCs) from established cell lines using sphere cultures was achieved efficiently without GFs. Enhanced sphere-forming activity was detected in GF-free cultures compared with GF-containing cultures in MCF7, A549 and U87 cells. Analysis of conditioned media (CM) demonstrated an elevated level of EGF secretion in GF-free CM compared with GF-containing CM in MCF7 cells. By contrast, cells in GF-free CM exhibited almost no secretion of bFGF, whereas cells in GF-containing CM secreted a high level of bFGF. The addition of EGF enhanced sphere formation, whereas bFGF suppressed sphere formation under GF-free conditions in MCF7 cells. Conversely, the addition of bFGF suppressed sphere formation in these cells. Notably, bFGF markedly suppressed EGF receptor (EGFR) expression and EGF secretion in MCF7 and A549 cells. Consistent with this result, EGFR blockade with pharmacological inhibitors significantly suppressed sphere formation in MCF7 and A549 cells under GF-free conditions. Furthermore, the neutralization of EGF also inhibited sphere formation, whereas bFGF neutralization still enhanced sphere formation under these conditions. Together, CSCs may be maintained in a serum-free culture condition without GFs, possibly through autocrine secretion of GFs such as EGF, and the addition of bFGF may not be sufficient for the enrichment of stem-like cancer cells.