A major complication with enzyme replacement therapy of Factor VIII (FVIII) in Hemophilia A (HA) is the development of anti-drug antibodies. Recently, we have shown that FVIII administration in the presence of heterogeneous phosphatidylserine (PS) nanoparticles derived from a natural source induces tolerance to FVIII, suggesting that PS converts an immunogen to a tolerogen. However, the specific structural features responsible for the immune-regulatory properties of PS is unclear. Identifying a specific PS species that is responsible is critical in order to further develop and optimize this nanoparticle. Further, clinical development of this lipid-based strategy requires optimization of the lipid particle that is homogeneous and synthetic. Here, we investigate the ability of mono-acylated Lyso-PS to induce hypo-responsiveness towards FVIII in HA mice. Administration of both PS and Lyso-PS FVIII significantly reduced anti-FVIII antibody responses despite rechallenge with FVIII. Additionally, the Lyso-PS-mediated effect was shown to be antigen-specific as mice responded normally against a rechallenge with an unrelated antigen, ovalbumin. Furthermore, the hypo-responsiveness observed with Lyso-PS may involve interactions with a specific PS receptor, TIM-4, along with increasing regulatory T-cells. These data indicate that using Lyso-PS allows for a more homogenous formulation in order to induce tolerance towards therapeutic proteins.