FHL2, a member of the four and one half LIM domain protein family, is a critical transcriptional modulator.Here, we identify FHL2 as a critical regulator of hematopoietic stem cells (HSCs) that is essential for maintaining HSC self-renewal under regenerative stress.We find that Fhl2 loss has limited effects on hematopoiesis under homeostatic conditions.In contrast, Fhl2-null chimeric mice reconstituted with Fhl2-null bone marrow cells developed abnormal hematopoiesis with significantly reduced numbers of HSCs, hematopoietic progenitor cells (HPCs), red blood cells and platelets as well as hemoglobin levels.In addition, HSCs displayed a significantly reduced self-renewal capacity and were skewed toward myeloid lineage differentiation.We find that Fhl2 loss reduces both HSC quiescence and survival in response to regenerative stress, probably as a consequence of Fh12-loss-mediated down-regulation of cyclin dependent kinase (CDK)-inhibitors, including p21 (Cip), p27(Kipl) and p57(Kip2).Interestingly, FHL2 is regulated under control of a tissue specific promoter in hematopoietic cells and it is down-regulated by DNA hypermethylation in leukemia cells.Furthermore, we find that down-regulation of FHL2 frequently occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, raising a possibility that FHL2 down-regulation plays a role in the pathogenesis ofmyeloid malignancies.