Nestin knockout leads to embryonic lethality and self-renewal deficiency in neural stem cells (NSCs). However, how nestin maintains self-renewal remains uncertain. Here, we used the dosage effect of nestin in heterozygous mice (Nes+/−) to study self-renewal of NSCs. With existing extracellular signaling in vivo or in vitro, nestin levels do not affect proliferation ability or apoptosis when compared between Nes+/− and Nes+/+ NSCs. However, self-renewal ability of Nes+/− NSCs is impaired when plated at a low cell density and completely lost at a clonal density. This deficiency in self-renewal at a clonal density is rescued using a medium conditioned by Nes+/+ NSCs. In addition, the Akt signaling pathway is altered at low density and reversed by conditioned medium. Our data show that secreted factors contribute toward maintaining self-renewal of NSCs by nestin, potentially through Akt signaling.