Chromodomain helicase DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin-remodeling factor that is encoded by the most frequently mutated gene in individuals with autism spectrum disorder. CHD8 is expressed not only in neural tissues but also in many other organs; however, its functions are largely unknown. Here, we show that CHD8 is highly expressed in and maintains the stemness of hematopoietic stem cells (HSCs). Conditional deletion of Chd8 specifically in mouse bone marrow induces cell cycle arrest, apoptosis, and a differentiation block in HSCs in association with upregulation of the expression of p53 target genes. A colony formation assay and bone marrow transplantation reveal that CHD8 deficiency also compromises the stemness of HSCs. Furthermore, additional ablation of p53 rescues the impaired stem cell function and differentiation block of CHD8-deficient HSCs. Our results thus suggest that the CHD8-p53 axis plays a key role in regulation of the stemness and differentiation of HSCs.Keywords: CHD8; apoptosis; bone marrow transplantation; cell cycle arrest; chromatin remodeler; colony formation assay; differentiation block; hematopoietic stem cell; p53; stemness.