The aortic valve maintains unidirectional blood flow between the left ventricle and the systemic circulation. When diseased, the valve is replaced either by a mechanical or a bioprosthetic heart valve that typically fail within ~15 years of implantation, necessitating the development of more structurally and biologically sufficient long-term replacements. Tissue engineering provides a possible avenue for development, combining cells, scaffolds, and biochemical factors to regenerate tissue or replace function. The overall goal of this dissertation was to create a foundation for the rational design of a tissue engineered aortic valve. The novel approach taken in this thesis research was to view each of the three valve leaflets as a laminate structure that could be modularly assembled into a tissue engineered valve leaflet. The first three aims consider the leaflet as a laminate structure comprising of layers of collagen, elastin, and glycosaminoglycans (GAGs). In the first aim, the effect of GAGs in the middle layer of the leaflet, the spongiosa, on the tensile properties and stress relaxation in the leaflet was investigated, by removing GAGs through increasing amounts of hyaluronidase (HAse). A decrease in GAGs led to significantly higher elastic moduli, ultimate tensile strengths, and hysteresis in the leaflet. In the second aim, the elastic fiber network of the leaflet, which is commonly found in the inflow layer, the ventricularis, but also found in the spongiosa, was characterized using immunohistochemistry and scanning electron microscopy. This structure was found to have regionally varying thicknesses and patterns. In the third aim, a novel hydrogel-fiber composite design was proposed to mimic the outflow layer of the leaflet, the fibrosa, which consists of highly aligned collagen fibers. This composite composed of aligned electrospun poly(?-caprolactone) (PCL) with a poly(ethylene glycol) diacrylate (PEGDA) matrix. Surface modification and embedding of the PCL did not significantly alter the anisotropy or strength of the underlying PCL scaffold, providing the basis for anisotropic, biocompatible scaffold. In the last aim, the approach taken was that the aortic valve was a layered structure of valvular endothelial cells (VEC) and interstitial cells (VIC). A novel co-culture model was designed using magnetic levitation. This technique was used to create co-culture models within hours, while maintaining cell phenotype and function, and inducing extracellular matrix formation, as shown by immunohistochemical stains and qRT-PCR results. The overall result of this dissertation is a clearer understanding of the layered structure-function relationship of the aortic valve, and its application towards heart valve tissue engineering.