Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis. Display omitted •MED12 is required for hematopoietic stem cell function in a cell-autonomous manner•Depleting other members of the Mediator kinase module does not affect HSC survival•MED12 deletion leads to H3K27Ac loss at enhancers of key HSC genes, such as c-Kit•MED12 cooperates with P300 at enhancers of essential hematopoietic genes Aranda-Orgilles et al. show in vivo a cell-autonomous requirement of MED12 in the maintenance of hematopoietic stem cell function independent of CYCLIN C/CDK8. MED12 cooperates with P300 to preserve enhancer activity, and loss of MED12 causes H3K27Ac depletion at enhancers of essential HSC genes and failure of hematopoietic-specific transcriptional programs.