Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other gamma-secretase substrates such as APP. LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (gamma-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other gamma-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of gamma-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the gamma-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other gamma-secretase substrate(s) along with Notch are important in the maintenance of the sternness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other gamma-secretase substrates during differentiation. (C) 2010 Elsevier Inc. All rights reserved.