In tissue engineering, endochondral ossification (EO) is often replicated by chondrogenically differentiating mesenchymal stromal cells (MSCs)in vitroand achieving bone formation throughin vivoimplantation. The resulting marrow-containing bone constructs are promising as a treatment for bone defects. However, limited bone formation capacity has prevented them from reaching their full potential. This is further complicated since it is not fully understood how this bone formation is achieved. Acellular grafts derived from chondrogenically differentiated MSCs can initiate bone formation; however, which component within these decellularised matrices contribute to bone formation has yet to be determined. Collagen type X (COLX), a hypertrophy-associated collagen found within these constructs, is involved in matrix organisation, calcium binding and matrix vesicle compartmentalisation. However, the importance of COLX during tissue-engineered chondrogenesis and subsequent bone formation is unknown. The present study investigated the importance of COLX by shRNA-mediated gene silencing in primary MSCs. A significant knock-down of COLX disrupted the production of extracellular matrix key components and the secretion profile of chondrogenically differentiated MSCs. Followingin vivoimplantation, disrupted bone formation in knock-down constructs was observed. The importance of COLX was confirmed during both chondrogenic differentiation and subsequent EO in this tissue engineered setting.