The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), H2O2 (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O2), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via pERK1/2 and pAKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content.Keywords:extracellular vesicle; microenvironment mimetic; production; stem cell.