<p>Biofilm formation and antibiotic tolerance are major contributors to the persistence of <i>Staphylococcus aureus</i> infections, yet how the host environment affects these phenotypes remains poorly understood. Here, we show that incubation in human serum primes <i>S. aureus</i> to form robust biofilms and tolerate vancomycin and daptomycin, last resort antibiotics for the treatment of antibiotic-resistant staphylococcal infections. Mechanistically, we demonstrate that the staphylococcal Geh lipase is essential for serum-induced biofilm formation by liberating glycerol from host lipids, which is then used to promote increased synthesis of D-alanylated wall teichoic acids, driving biofilm development. Inhibition of the Geh lipase or wall teichoic acid synthesis markedly reduces biofilm formation and restores antibiotic susceptibility, highlighting clinically achievable strategies to inhibit host-induced biofilm formation and prevent the associated antibiotic tolerance. Together, our findings reveal a host-driven mechanism of biofilm-associated antibiotic tolerance in <i>S. aureus</i> and provide rational targets for therapeutic intervention.</p>