We have recently shown that Natural Killer (NK) cells control survival and differentiation of Cancer Stem-like Cells (CSCs) through two distinct phenotypes of cytotoxic and anergic NK cells, respectively. In this report, brain CSCs and their serum and NK cell differentiated counterparts were studied. Serum-differentiated brain CSCs were significantly less susceptible to NK cells and CTL direct cytotoxicity as well as NK cell mediated Antibody Dependent Cellular Cytotoxicity (ADCC), whereas their CSCs were highly susceptible. The levels of CD44 and EGFR were higher in brain tumor CSCs when compared to the serum-differentiated tumors. No differences could be observed for the expression of MHC class I between brain tumor stem cells and their serum-differentiated counterparts. Moreover, supernatants from the combination of IL-2 and anti-CD16mAb treated NK cells (anergized NK cells) induced resistance of brain tumor CSCs to NK cell mediated cytotoxicity. Unlike serum-differentiated CSCs, NK supernatant induced differentiation and resistance to cytotoxicity in brain CSCs correlated with the increased expression of CD54 and MHC class I. The addition of anti-MHC class I antibody moderately inhibited NK mediated cytotoxicity against untreated or serum-differentiated CSCs, whereas it increased cytotoxicity against NK supernatant differentiated tumors. Therefore, two distinct mechanisms govern serum and NK supernatant mediated differentiation of brain tumors.