In the context of skeletal tissue development and repair, endochondral ossification has inspired a new approach to regenerate bone tissue in vivo using a cartilage intermediate as an osteoinductive template. The aim of this study was to investigate the behavior of mesenchymal stem cells (MSCs) in regard to in vitro cartilage formation and in vivo bone regeneration when combined with different three-dimensional (3D) scaffold materials, i.e., hydroxyapatite/tricalcium phosphate (HA/TCP) composite block, polyurethane (PU) foam, poly(lactic-co-glycolic acid)/poly(ε-caprolactone) electrospun fibers (PLGA/PCL) and collagen I gel. To this end, rat MSCs were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by in vivo subcutaneous implantation for 8 weeks. Nonetheless, the quality and maturity of in vivo ectopic bone formation appeared to be scaffold/material-dependent. Eight weeks of implantation was not sufficient to ossify the entire PLGA/PCL constructs, albeit a comprehensive remodeling of the cartilage had occurred. For HA/TCP, PU and collagen I scaffolds, more mature bone formation with rich vascularity and marrow stroma development could be observed. These data suggest that chondrogenic priming of MSCs in the presence of different scaffold materials allows the establishment of reliable templates for generating functional endochondral bone tissue in vivo without using osteoinductive growth factors. The morphology and maturity of bone formation.