Endochondral ossification is an indirect path of bone formation involving the condensation of progenitor cells into a cartilaginous anlage that grows in size, matures, and releases growth factors to promote blood vessel invasion and cartilage remodeling, resulting in a robust, vascularized bone organ. This is the dominant pathway of bone formation in both long bone development and fracture healing and has emerged as an attractive tissue engineering template to address the lack of angiogenesis and poor engraftment seen in current intramembranous engineering approaches. Though it is known that mechanical cues are essential to the progression of endochondral ossification in both processes, the role of mechanical loading in endochondral bone defect regeneration has not been investigated. In vivo, we applied mechanical loading to chondrogenically-primed hMSCs within a critically-sized rat femoral defect either immediately after implantation, or delayed until 4 weeks into chondrogenesis, compared to unloaded controls. Delaying loading significantly improved bone regeneration through increased bone volume, bridging across the defect, and recovery of mechanical properties compared to unloaded controls, and this was further potentiated by supplementation of additional growth factors. Conversely, early loading increased bone volume but did not enhance bridging or mechanical properties. There was significantly decreased vascular invasion and persistent unmineralized gaps of cartilage, resulting in lack of functional repair. This suggests that the timing of in vivo mechanical loading during progenitor cell maturation is an important factor in directing tissue formation. To this end, we investigated, in vitro, the role of endochondral priming length prior to dynamic compression in a custom-made bioreactor. Mechanical load initiated chondrogenic gene expression at all stages, but only enhanced endochondral progression when applied after 6 weeks of priming. Loading at earlier stages of priming had an inhibitory effect on chondrocyte maturation, consistent with in vivo findings. Together these results suggest that mechanical loading is necessary to induce successful endochondral progression for bone tissue engineering.