The prosthetic valves available to treat heart valve disease are poorly suited for younger and more active patients. Tissue engineering has tremendous potential for impact here by providing a living de novo graft capable of needed growth and integration. However, despite over 20 years of research, none are clinically ready. Recent efforts have focused on transcatheter delivery, which even if successful targets the very old and is not suitable for the young. We have pioneered 3D tissue printing technology to create anatomically accurate and mechanically heterogeneous fully cellularized living heart valve conduits. First, we have developed printable (extrudable), photocrosslinkable, biologically derived, cell-friendly hydrogel building blocks that attain mechanical properties of the root sinus and leaflets. Second, we have optimized the engineering control of soft tissue hydrogel printing, achieving a 200 μm minimum extrusion width and spatial precision of 15 μm. Third, we have developed and integrated an on-board photocrosslinking ultraviolet light-emitting diode (UV-LED) that rapidly cures tissues simultaneous with printing. Fourth, we have developed algorithms that process 3D image datasets of a native valve (e.g. CT or MRI) to automatically extract the valve geometry and segregate root and leaflet regions. These innovative technologies enable precise design of conduit macro-geometry to affect native-like valve tissue formation. We have further synthesized and characterized unique formulations of PEGDA, Alginate, gelatin, and hyaluronic acid, individually and blended, that are 3D printable. We have optimized formulations to mimic the physiological biomechanical range of human pediatric valves, and successfully fabricated cell-seeded 6-month-old sized valves. We have further demonstrated tunable stiffness (via altering molecular weights and blending), cell adhesion ligand density (via covalent conjugation of adhesion sequences like RGDS), and growth factor tethering. We recently showed that these components help maintain native root wall smooth muscle cell (SMC) and VIC phenotype in 3D culture. We also show that stiffness and adhesion ligand density control mesenchymal stem cells (MSC) differentiation towards these critical lineages for TEHV and away from calcification. We have also created a vector-based toolpath creation algorithm for achieving local variation in printed tissue composition with a small number of components. We believe these heterogeneous anatomically precise components are essential to improving the success of heart valve tissue engineering.