During the brain development, the process of neural stem cells (NSCs) proliferation and differentiation is precisely regulated. The deficiency in the embryonic brain development will cause serious developmental disorders. Epigenetic modifications play critical roles in controlling proliferation and differentiation in different types of stem cells. Histone variants, as one of epigenetic regulators, have been reported to be associated with many bioprocesses. Among different variants, H3.3 is one of the important epigenetic regulators, but its role in embryonic NSCs remains unclear. Here we demonstrate that H3.3 is intrinsically required for NSCs proliferation and differentiation. Suppression of the H3.3 mediated by shRNAs causes the reduction of the PAX6-positive NSCs proliferation, and promotes the premature terminal mitosis and neuronal differentiation. Particularly, the level of the H4K16ac is selectively reduced in the H3.3 knockdown NSCs. We further confirm that H3.3 is directly interacted with the MOF, a specific H4K16 acetyltransferase. Interestingly, H3.3/MOF increases the level of H4K16ac by a mutual cooperation manner. However, the H3.3K36R mutant could not increase the level of H4K16ac. RNA-seq data show the GLI1, a transcriptional regulator, is downregulated in H3.3 knockdown NSCs. Furthermore, the neurogenesis phenotype of the GLI1 knockdown is consistent with the H3.3 knockdown. Overexpression of the H3.3, MOF, and GLI1 could rescue the abnormal phenotype caused by H3.3 knockdown in the embryonic brain, but H3.1 or H3.3K36R overexpression can not rescue it. Taken together, these results suggest that H3.3 cooperates with MOF to increase the level of the H4K16ac and the GLI1, and then regulates the NSCs proliferation and differentiation.