Calcific aortic valve disease (CAVD) is an active, cell-mediated pathology associated with significant cardiovascular morbidity but with no current pharmacologic treatments. In late CAVD, the aortic valve (AV) leaflets become fibrotic and calcified, but early CAVD is characterized by proteoglycan-rich subendothelial lesions in the fibrosa layer of the leaflets. Valve interstitial cells (VICs) in these early lesions express higher levels of genes associated with bone and cartilage formation in situ, motivating the hypothesis that the lesion microenvironment promotes VIC pathological gene expression. VIC pathological differentiation has been shown to be affected by both ECM mechanics and composition, but the relevance of these in vitro studies to the lesion microenvironment was unknown. I first determined if early AV lesions have local mechanical properties that are distinct from those normal valve tissue. A custom microindentation device was developed and validated. For the first time, I showed that the compressive moduli of early lesions were ~2.4 fold softer than healthy non-lesion regions. As a top-down approach to test the effect of the lesion microenvironment on VIC responses, I then established a novel ex vivo decellularized valve model of normal valve ECM and early CAVD, defining decellularization conditions that maximally removed cells and preserved ECM microenvironment. I then applied the model to investigate the influence of normal versus diseased ECM on VIC phenotypic expression. Several genes involved in osteogenic (e.g., RUNX2, MSX2 and SPP1) and chondrongenic (e.g., ACAN, HYAL2 and CHSY1) processes were upregulated in naïve VICs grown in lesion microenvironments compared to non-lesion fibrosa areas. Inflammatory and ECM related genes were differentially expressed between lesion and non-lesion fibrosa. These in vitro expression patterns match lesion vs. non-lesion gene expression profiles measured in vivo, supporting the validity of my decellularized tissue model and a role for the ECM in driving VIC gene expression associated with pathologic differentiation. The significant findings of this thesis provide novel insights into the role the ECM plays in regulating native valve (dys)function. This thesis also contributes new tools and knowledge to disease modeling research, with broad applications towards better understanding the role of the ECM in health and disease.