The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating haematopoietic stem cell (HSC) function in adult haematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to haematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs) and the decreased numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages. Loss of HBO1 resulted in an abnormally high rate of recruitment of HSCs into the cell cycle, leading to the exhaustion of the HSC pool. Whole-genome transcriptome analysis revealed that genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek (Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9. Epigenomic profiling of HSC-enriched cell populations using CUT&Tag further revealed that HBO1 was required for H3K14Ac throughout the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult haematopoiesis.