Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification;​ however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.