Human Hematopoietic Stem/Progenitor Cells (HSPC) mainly reside in the bone marrow (BM) but few circulating cells (cHSPC) are found in peripheral blood (PB) and their amount can be increased by mobilizing agents for gene therapy (GT) purposes. Combining phenotypic and functional characterizations with single-cell transcriptome profiling and integration site analyses, we are studying the behavior of human resident, circulating and mobilized. By evaluating the reconstitution kinetic after GT in Wiskott-Aldrich Syndrome patients we found that mobilized peripheral blood(MPB)-HSPC allowed faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage, as compared to BM HSPC. This correlated with higher amount of myeloid and primitive progenitors contained in MPB HSPC. Finally, BM- and MPB-GT patients display distinct kinetics of hematopoietic recovery, but similar long-term reconstitution. We assessed HSPC mobilization kinetics in patients with different inherited disorders as well as adult healthy donors(HD) undergoing mobilization with G-CSF (G) with or without Plerixafor (G+P). Primitive and myeloid progenitors showed the highest mobilization capability after G, while all HSPC mobilized after administration of P. This subset-specific mobilization propensity after G inversely correlated with HSPC CXCR4 expression. By reproducing patients' mobilization protocol in xenotransplanted mice we are assessing the impact of CXCR4 expression on the distinct HSPC mobilization kinetics observed. Finally, we found that cHSPC and BM-HSPC display distinct compositions but comparable BM homing and differentiation potential after xenotransplantation. Moreover, the number of both total and primitive HSPC subsets circulating in PB is high in young infants, suggesting the possible exploitation of cHSPC as alternative stem cell source. We are currently investigating the molecular mechanisms of HSPC trafficking and evaluating the capability of cHSPC to be transduced and expanded in vitro.