The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development through its role in H3K14 acetylation at gene loci critical for embryonic development. In this study, we report the role of HBO1 in regulating haematopoietic stem cell (HSC) function in adult haematopoiesis. We used two complementary conditional knockout models (Mx1-Cre and Rosa26-CreER). Hbo1 null mice became moribund due to haematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Competitive transplantation of Hbo1 deleted bone marrow cells failed to repopulate in haemoablated recipients. Hbo1 deletion caused a rapid loss of haematopoietic progenitors (HPC). The numbers of lineage-restricted progenitors for erythroid, myeloid, B-cell or T-cell lineage were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of HSCs into the cell cycle. The cycling HSCs produced progenitors at the expense of self-renewal, which led 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. This included genes known to be essential for HSC quiescence and self-renewal, as well as genes important for lineage-specific progenitor cells. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSCs. Altogether, our findings establish that HBO1 is essential for HSC quiescence, self-renewal and lineage differentiation in adult haematopoiesis.