Introduction: Biomaterials such as autologous platelets have recently gained intense research interest (1). However, the mechanisms regarding platelet mediated skeletal myogenesis remain to be established. The aim of this study was to determine the role of platelet releasate in skeletal myogenesis and muscle stem cell fate in vitro and ex vivo and in vivo. Methods: The effects of platelet releasate on proliferation and differentiation of skeletal myoblasts by means of cell proliferation assays, immunohistochemistry, gene expression and cell bioenergetics were assessed. We expanded in vitro findings on single muscle fibres by determining the effect of platelet releasate on murine skeletal muscle stem cells ex vivo using protein expression profiles for key myogenic regulatory factors. Most importantly, we validated our findings on an in vivo model of acute muscle injury via cardiotoxin. Statistical analysis was performed by one way ANOVA followed by Tukey’s post hoc test. Results: We have shown that platelet releasate dose dependently stimulates myoblast proliferation and temporally stimulates differentiation via upregulation of MyoD and an increase in muscle stem cell commitment to differentiation. Proteomics revealed that the abundance of PDGF and VEGF ligands varies according to the platelet agonist used. Inhibiting these ligand binding sites dose dependently inhibited myoblast proliferation. Skeletal muscle stem cell markers for ex vivo proliferation and cell fate lineage (Cyclin D1 and Scrib, respectively) were robustly upregulated when exposed to platelet releasate. Most importantly, the translational aspect of platelet releasate to an in vivo model promoted myogenic regeneration. We have shown novel data that supra physiological levels of the platelet secretome can further increase myoblast proliferation in a dose dependent manner. Conclusion: Platelet releasate is a powerful driver of skeletal myoblast proliferation (2). This study provides novel mechanistic insights on the role of platelet releasate in skeletal myogenesis by directing muscle stem cell fate and sets the physiological basis for exploiting platelets as biomaterials in regenerative medicine.