Hematopoietic cell transplantation (HCT), dependent on hematopoietic stem cells (HSC), is widely used for treatment of many hematological and nonhematological disorders [1]. Successful clinical outcome of HCT predominantly relies on adequate homing and long-term engraftment of HSC into the bone marrow (BM). Enhancing the efficacy of HSC homing and engraftment could have a relevant impact on improving HCT and patient survival, especially when limiting numbers of HSCs are available, as in using umbilical cord blood (CB) or poorly mobilized peripheral blood [2–4]. Although efforts have been devoted to developing potential means to enhance HSC engraftment, including boosting HSC expansion [5, 6], enhancing HSC homing [7, 8], mitigating extra physiologic oxygen shock/stress [9], or generating HSCs by reprogramming [10, 11], it is not yet clear which procedures will have the most clinical efficacy, and there is still an urgent need in clinical practice for simple and efficacious methods to enhance HSC engraftment. Nitric oxide (NO) is a gaseous molecule which acts as a signal molecule in mammalian cells, and plays important roles in a variety of physiological regulations, such as synaptic plasticity, endothelial cell relaxation, and immune responses [12, 13]. NO can freely diffuse across the cellular membrane and activate a cytoplasm enzyme, soluble guanylyl cyclase (sGC), to produce cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP acts as an important secondary messenger and activates cGMP-dependent protein kinase (PKG) to regulate a broad spectrum of downstream processes. In hematopoietic system, NO signaling is necessary for proper differentiation of HSC and lineage commitment involving multiple signaling pathways [14]. Besides, NO signaling is required in vascular niche for HSC generation and production during embryogenesis [15], implicating potential beneficial effects of NO signaling for HSC biology and HCT. However, potential roles of NO and cGMP in regulating HSC homing and engraftment remain to be determined. To explore the role of NO signaling in regulating HSC engraftment, effects of NO signaling activators on human CB HSC engraftment in NSG (NOD. Cg-PrkdcscidIL2rgtm1Wjl/Sz) mice were evaluated, which is the widely accepted golden standard for in vivo determination of human HSC functionality. We first used sodium nitroprusside (SNP, an NO donor), and Riociguat (a sGC stimulator) to activate NO signaling. CD34+ cells were isolated from fresh human CB, and treated with SNP or Riociguat for 16h in medium