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Split thickness skin grafts (STSGs) are one of the standard treatments available for full thickness wound repair when full thickness grafts (FTGs) are not viable, such as in the case of wounds with large surface areas. The donor sites of STSGs may be harvested repeatedly, but STSG transplants are still limited by insufficient blood supply at the early stages of wound healing. Prevascularized human mesenchymal stem cell (hMSC) sheets may accelerate wound healing and improve regeneration by providing pre-formed vessel structures and angiogenic factors to overcome this limitation. This book chapter provides the protocol of co-culturing hMSCs and endothelial cells to attain a prevascularized hMSC cell sheet (PHCS). The protocols for implantation of the prevascularized stem cell sheet for full thickness skin wound repair in a rat autologous skin graft model as well as the evaluation of the wound healing effects are also provided.