The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin⁻Sca-1 ⁺c-Kit ⁺ (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1^−/−, Pim2^−/−, Pim3^−/− single knockout (KO) mice. HSCs from Pim1^−/− KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2^−/− or Pim3^−/− KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34⁻ HSCs was reduced by approximately 28-fold in Pim1^−/− KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.