Adult stem cells are a promising tool to positively influence bone regeneration. Concentrated bone marrow therapy entails isolating osteoprogenitor cells during surgery with, however, only low cells yield. Two step stem cell therapy requires an additional harvesting procedure but generates high numbers of progenitor cells that facilitate osteogenic pre-differentiation. To further improve bone regeneration, stem cell therapy can be combined with growth factors from platelet rich plasma (PRP) or its lysate (PL) to potentially fostering vascularization. The aim of this study was to investigate the effects of bone marrow concentrate (BMC), osteogenic pre-differentiation of mesenchymal stromal cells (MSCs), and PL on bone regeneration and vascularization. Bone marrow from four different healthy human donors was used for either generation of BMC or for isolation of MSCs. Seventy-two mice were randomized to six groups (Control, PL, BMC, BMC + PL, pre-differentiated MSCs, pre-differentiated MSCs + PL). The influence of PL, BMC, and pre-differentiated MSCs was investigated systematically in a 2 mm femoral bone defect model. After a 6-week follow-up, the pre-differentiated MSCs + PL group showed the highest bone volume, highest grade of histological defect healing and highest number of bridged defects with measurable biomechanical stiffness. Using expanded and osteogenically pre-differentiated MSCs for treatment of a critical-size bone defect was favorable with regards to bone regeneration compared to treatment with cells from BMC. The addition of PL alone had no significant influence; therefore the role of PL for bone regeneration remains unclear.