Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth.