Calcific aortic valve disease (CAVD) is the most common valvular heart disease and lacks effective pharmacological treatment in large part because the molecular mechanisms underlying the disease are not well understood. CAVD lesions often include osteoblasts and form preferentially in the natively stiffer fibrosa side of the leaflet, suggesting roles for valve interstitial cell (VIC) osteogenesis and matrix biomechanics in valve disease. In other cell types, osteogenic differentiation is mediated by four and a half LIM domains protein 2 (FHL2) and modulated by matrix stiffness via RhoA, however their roles in mechanically regulated VIC osteogenesis and valve disease are unknown. This thesis provides the first examination of FHL2 expression in human aortic valves from CAVD patients and found upregulated FHL2 expression in diseased regions co-localized with elevated levels of the osteogenic marker Runx2 suggesting FHL2 could play a role in disease. In vitro experiments utilizing substrates of different stiffness found RhoA activation and FHL2 nuclear localization were mechanoresponsive in VICs, increasing as matrix stiffness increased. Increased matrix stiffness and RhoA activation also promoted VIC osteogenic aggregation. Notably, FHL2 knockdown inhibited aggregate formation downstream of RhoA highlighting FHL2 as an important mediator of mechanically regulated VIC osteogenesis. The role of this mechanosensitive FHL2 pathway was examined in vivo with Ldlr-/-;​Apob^100/100 mice that had mild CAVD as determined by elevated transvalvular velocities and aortic velocity ratios. FHL2 and Runx2 expression in the murine aortic valves were co-localized consistent with the human results. A protocol was developed that optimized sectioning technique, tissue rehydration, and probe diameter to allow the measurement of physiologically relevant stiffness in murine aortic valves by atomic force microscopy (AFM) at cellular length-scales and increased FHL2 expression in cells correlated with increased local matrix stiffness supporting the in vitro results. The work in this thesis provides additional characterization of the Ldlr-/-;​Apob^100/100 mouse model of CAVD, presents a refined AFM technique to spatially correlate local matrix stiffness measures with cellular protein expression to enable ex vivo mechanotransduction studies, and identifies a mechanically regulated FHL2 signaling pathway that directs VIC osteogenesis in vitro providing insight into CAVD development and potential therapeutic targets.