smac mimetic promotes glioblastoma cancer stemlike cell differentiation by activating nfb recently a broader role of inhibitor of apoptosis iap proteins besides their antiapoptotic functions has been described therefore we investigated the effect of nontoxic concentrations of the smallmolecule smac mimetic bv6 which antagonizes iap proteins on differentiation of cancer stemlike cells cslcs derived from primary glioblastoma gbm specimens here we identify a novel function of bv6 in regulating differentiation of gbm cslcs by activating nfb bv6 at nonlethal doses stimulates morphological changes associated with the differentiation of gbm cslcs bv6 increases transcriptional activity mrna and protein levels of the astrocytic marker gfap without altering expression of the neuronal marker iiitubulin indicating that bv6 induces astrocytic differentiation of gbm cslcs molecular studies reveal that bv6 triggers processing of the nfb subunit p100 to p52 nuclear translocation of p52 and p50 and increased nfb dnabinding intriguingly inhibition of nfb by overexpression of dominantnegative ib superrepressor ibsr blocks the bv6stimulated increase in gfap and differentiation interestingly this bv6stimulated differentiation is associated with reduced expression of stemness markers such as cd133 nanog and sox2 in gbm cslcs in contrast bv6 does not alter cell morphology differentiation and expression of stemness markers in nonmalignant neural stem cells importantly bv6 treatment reduces clonogenicity of gbm cslcs in vitro and in vivo suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice by identifying a novel role of bv6 in promoting differentiation of gbm cslcs these findings provide new insights into smac mimeticregulated nonapoptotic functions with important implications for targeting gbm cslcs