Calcific aortic valve disease (CAVD) is characterized by the thickening and calcification of aortic valve leaflets. CAVD is a multifaceted disease caused by aberrant differentiation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs) in response to changes in physiological mechanical environments. VECs can undergo endothelial-to-mesenchymal transition (EndMT) and contribute to the resident pool of fibroblast cells (VICs) within the aortic valve and upon stimulation, VICs can differentiate into myofibroblast- or osteoblast-like cells. The Wnt/β-catenin signaling pathway has been implicated in CAVD, VIC myofibrogenic, and osteogenic differentiation, however, its roles in mechanically-regulated EndMT and VIC osteogenic differentiation have not been investigated. The first part of this thesis examines the role of matrix stiffness and β-catenin in EndMT of VECs and found that TGF-β1 induced EndMT in a stiffness- and β-catenin-dependent manner. Notably, the initiation of EndMT (disruption of cell morphology and membrane VE-cadherin expression) was insensitive to matrix stiffness, but α-SMA fiber formation and nuclear translocation of β-catenin both increased on stiff substrate. Endostatin treatment revealed that β-catenin was necessary for TGF-β1-induced transformation of VECs to α-SMA positive myofibroblasts, and this association was confirmed by immunofluorescent staining of sclerotic aortic valves. The second part of the thesis analyzed the role of β-catenin, FoxO1, and cyclic stretch on VIC osteogenic differentiation. We demonstrated for the first time that FoxO1 expression is increased and co-localized with Runx2 in calcified region of diseased human aortic valve using immunostaining. In vitro experiments using siRNA knockdown and exogenous overexpression confirmed the causal effect of FoxO1 on the expression of osteogenic genes RUNX2, ENPP1, and SPP1 in VICs. Surprisingly, β-catenin knockdown had no effect on VIC osteogenic gene expression and thus was unlikely to be a co-factor of FoxO1 in regulating VIC osteogenic differentiation. Although short-term mechanical stretch had no effect on FoxO1, β-catenin, and osteogenic gene expression, long-term stretch promoted dystrophic calcific nodule formation. The findings in this thesis characterized the role of Wnt/β-catenin signaling pathway in mechanically-regulated EndMT and VIC osteogenic differentiation, identified FoxO1 as a novel mediator of VIC osteogenic differentiation, and provided insights into the molecular mechanisms behind CAVD pathogenesis and potential therapeutic targets.