The delivery of osteogenic factors is a proven therapeutic strategy to promote bone regeneration. Bone morphogenetic proteins (BMPs) constitute a family of cytokines with well-known osteogenic and bone regenerative abilities. However, clinical uses of BMPs require high doses that have been associated with complications such as osteolysis, ectopic bone formation, or hematoma formation. In the present work, we sought to improve bone tissue engineering through an approach that combines the use of bone marrow-derived mesenchymal stem cells (BMMSCs), composite scaffolds, and osteoinductive agents. We employed a composite gelatin/CaSO4 scaffold that allows for an early expansion of seeded BMMSCs, which is followed by an increased level of osteogenic differentiation after 10 days in culture. Furthermore, this scaffold enhanced bone formation by BMMSCs in a mouse model of critical-sized calvarial defect. More importantly, our results demonstrate that ex vivo pretreatment of BMMSCs with low amounts of BMP-2 (2 nM) and Wnt3a (50 ng/mL) for 24 h cooperatively increases the expression of osteogenic markers in vitro and bone regeneration in the critical-sized calvarial defect mouse model. These data provide a strong rationale for the development of an ex vivo cooperative use of BMP-2 and Wnt3a. Osteogenic factor cooperation might be applied to reduce the required amount of growth factors while obtaining higher therapeutic effects.