Mechanical factors play a critical role in the development, maintenance and repair of skeletal tissues. Mechanical stimulation can alter the course of healing by directing the differentiation of mesenchymal progenitor cells into the cells that form the various skeletal tissues, and can enhance or impair the repair of orthopaedic injuries. Several mechanoregulatory hypotheses describing the relationships between mechanical stimuli and skeletal tissue differentiation have been proposed;​ however, these hypotheses have not been fully tested, nor have the underlying mechanisms been established. Identification of the specific mechanical stimuli and molecular mechanisms that direct the differentiation of mesenchymal progenitor cells would provide insight for treating injuries. The focus of this dissertation was to further our understanding of the mechanobiology of skeletal tissue differentiation by identifying the mechanisms that regulate the differentiation of mesenchymal progenitor cells. The first part of this dissertation identified consistent associations between the patterns of the formation of skeletal tissues (bone, cartilage, fibrocartilage and fibrous tissues) and the magnitudes of strains (shear and principal strains) in a mechanically-stimulated bone defect in vivo. The second part of this dissertation found evidence that the Rho-family GTPases, as well as adhesion receptors and their associated focal adhesion proteins, may be possible mediators of the mechanotransduction mechanisms involved in the decisions of cell fate of the mesenchymal progenitor cells within the stimulated tissues. Finally, in an anticipation of the next steps in research on mechano-regulation of tissue differentiation, a microindentation technique was developed to determine the poroelastic material properties of the soft tissues forming in the callus. The values of Young’s modulus, Poisson’s ratio and permeability of articular cartilage were measured at the microscale and compared to those determined using standard macroscale techniques. Together, the findings of this dissertation further our understanding of the mechanoregulation of skeletal tissue differentiation, and can be used to inform and improve the various hypotheses regarding the mechanoregulation of tissue differentiation. Clinically, these results could potentially direct the development of therapies to improve treatment outcomes and reduce recovery time.