As the transcriptional coactivator CITED2 (CBP/p300-interacting-transactivator-with-an ED-rich-tail 2) can be overexpressed in acute myeloid leukemia (AML) cells, we analyzed the consequences of high CITED2 expression in normal and AML cells. CITED2 overexpression in normal CD34(+) cells resulted in enhanced hematopoietic stem and progenitor cell (HSPC) output in vitro, as well as in better hematopoietic stem cell (HSC) engraftability in NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. This was because of an enhanced quiescence and maintenance of CD34(+)CD38(-) HSCs, due in part to an increased expression of the cyclin-dependent kinase inhibitor CDKN1A. We demonstrated that PU.1 is a critical regulator of CITED2, as PU.1 repressed CITED2 expression in a DNA methyltransferase 3A/B (DNMT3A/B)-dependent manner in normal CD34(+) cells. CD34(+) cells from a subset of AML patients displayed higher expression levels of CITED2 as compared with normal CD34(+) HSPCs, and knockdown of CITED2 in AML CD34(+) cells led to a loss of long-term expansion, both in vitro and in vivo. The higher CITED2 expression resulted from reduced PU.1 activity and/or dysfunction of mutated DNMT3A/B. Collectively, our data demonstrate that increased CITED2 expression results in better HSC maintenance. In concert with low PU.1 levels, this could result in a perturbed myeloid differentiation program that contributes to leukemia maintenance.Leukemia advance online publication, 30 September 2014; doi:10.1038/leu.2014.259.