Heart valve leaflets are exposed to a continual barrage of complex mechanical forces that cannot be ignored when studying their biology. Tissue stiffness, cyclic tissue stretch and fluid shear stress affect valve endothelium and interstitial cells in unique and shared ways. We develop and apply unique in vitro bioreactor test beds to evaluate these interactions in 3D in vitro culture and in situ. We developed a unique mechanical analysis system that interrogates subcellular cellular and matrix fiber kinematics in live mouse valve leaflets. We identified that wildtype mouse mitral valves stiffened with age until 4 months, associated with increases in collagen content, fiber alignment, and cellular alignment. Older mitral valves (>10 months) exhibited decreased stiffness associated with disruption in collagen architecture. Interestingly, we determined that Fbn1C1039/+ murine mitral valves (the genetic mutation that causes Marfan syndrome) exhibit reduced stiffness and increased matrix disruption compared to wildtype, indicative of an immature tissue phenotype. We further decipher how the directionality (anisotropy) of mechanical loading affects valvular signaling independently of magnitude or frequency. We discovered that anisotropic strain is a potent driver of cellular reorganization and matrix remodeling. Furthermore, anisotropic strain can uniquely activate signaling activity and affect cell phenotype based on their orientation to the strain field. We also elucidate the mechanotransduction pathways involved, which will open a new path for treating valve disease through restoration of cellular mechanosensitivity.