The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics. •Kat6b gene deletion causes a reduction of the hematopoietic stem cell (HSC) pool•Kat6b gene deletion impairs the ability of HSCs to repopulate a transplant recipient•Kat6b gene deletion reduces H3K9 acetylation and expression of HSC genes•Sorted HSCs from Kat6a+/−Kat6b+/− mice lack competitive long-term repopulating capabilities In this article, Thomas and colleagues show that the histone acetyltransferase KAT6B has an essential role in fetal hematopoietic stem cells. They show that loss of KAT6B results in reduced expression of essential stem cell genes and loss of HSC activity. In addition, they show KAT6B acts together with a related protein KAT6A to regulate gene expression in HSCs.