Controlling the formation of new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, little is known about the molecular regulation that controls lymphatic tube formation in a synthetic matrix. In this study, we utilize hyaluronic acid (HA)-hydrogels to design a novel platform for decoupled investigation into how mechanical and biochemical cues regulate lymphatic vessel formation in a synthetic matrix. Using HA and controlling the degree of modification provides a method to preserve and modulate key lymphatic markers Prox1, LYVE-1, and Pdpn. The chemistry of the system allows for spatial and temporal patterning of specific peptides and substrate stiffnesses, and an MMP-sensitive crosslinker allowed cells to degrade and remodel their matrix. Through systematic optimization of multiple parameters, we have designed a system that allows human lymphatic endothelial cells (LECs) to self-assemble into vessels in vitro within 3 days. These engineered vessels can be cultured for up to 3 weeks and can be used for high-throughput mechanistic studies, or can be implanted into immunodeficient mice where they have demonstrated the ability to integrate and mature. Collectively, these studies report a novel, fully-defined 3D synthetic matrix system capable of generating lymphatic vessels in vitro that provide promise as an in vitro screening platform and as a therapeutic vessel transplant, which to our knowledge, is the first ever 3D lymphatic tissue engineering approach to not require the use of support cells.