The Haematopoietically-expressed homeobox gene, Hhex, is a transcription factor that is important for the maturation and proliferation of definitive haematopoietic progenitors and B-cell development during embryonic haematopoiesis12 . However, whether Hhex plays similar roles in adult haematopoiesis is currently unknown. To study this we employed an Hhex Inducible Knock-Out (iKO) mouse. Following Hhex deletion using Mx-Cre, mice displayed normal long-term myeloid cell development. However competitive serial transplants of iKO donor Bone Marrow (BM) revealed a defect in Haematopoietic Stem Cell (HSC) self-renewal with a progressive reduction in contribution of Hhex-null HSCs to haematopoiesis. Although, Hhex-null donor BM contained normal numbers of haematopoietic progenitors, there was a striking absence of blast colony recloning in vitro. Thus, whilst Hhex is dispensable for normal steady state myeloid development, it plays crucial roles in the self-renewal of HSCs in vitro and in vivo. To study the response of Hhex-null HSCs to haematopoietic stress, we performed sub-lethal myelo-ablation of Hhex iKO mice. This demonstrated a dramatically impaired ability of the HSCs to regenerate the myeloid compartment. RNAseq analysis of Hhex iKO LSK cells, verified by RT-PCR, showed that the deletion of Hhex lead to altered expression of cell cycle regulators, including a reduction in expression of Cyclin D1. These results demonstrate that Hhex is a key transcriptional mediator of HSC self-renewal and stress haematopoiesis and suggest it mediates these effects via regulation of cell cycling.