We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte–colony-stimulating factor (G-CSF)–induced mobilization of CD34⁺ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33⁺ myeloid and CD14⁺ monocytic cells, whereas mobilized CD34⁺ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF–treated donors. c-suPAR was able to chemoattract CD34⁺ KG1 leukemia cells and CD34⁺ HSCs, as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR_84-95). uPAR_84-95 induced CD34⁺ KG1 and CD34⁺ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition, uPAR_84-95 inhibited CD34⁺ KG1 and CD34⁺ HSC in vitro migration toward the stromal-derived factor 1 (SDF1), thus suggesting the heterologous desensitization of its receptor, CXCR4. Finally, uPAR_84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34⁺ HSCs. Our findings demonstrate that G-CSF–induced upregulation of uPAR on circulating CD33⁺ and CD14⁺ cells is associated with increased uPAR shedding, which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization.