Supported lipid bilayers (SLBs) have been used extensively in a variety of biotechnology applications and fundamental studies exploring lipid behavior. Despite their widespread use, various physicochemical parameters have yet to be thoroughly investigated for their impact on SLB formation. In this work, we have studied the importance of flow in inducing the rupture of surface adsorbed chicken egg-derived l-α-phosphatidylcholine (egg PC) vesicles on silica and gold surfaces via quartz crystal microbalance with dissipation monitoring (QCM-D). On silica at 25 °C, egg PC vesicles were found to adsorb in a flattened configuration (∼13 nm thick, compared to bulk vesicle diameters of ∼165 nm) but only undergo a transition to a stable SLB under flow conditions. In the absence of flow, an increase in system temperature to 37 °C was able to promote vesicle rupture and SLB formation on silica with a 10 times lower rupture time, compared to rupture under continuous flow (175 μL/min flow rate). Gold surfaces, with their increased hydrophobicity, led to less vesicle flattening once adsorbed (structures ∼60 nm thick), and did not support vesicle rupture or SLB formation, even at flow rates of up to 650 μL/min. We also showed that, under continuous flow conditions, vesicle adsorption rates on silica surfaces follow Langmuir kinetics, with an inverse dependence on bulk vesicle concentration, while an empirical power law dependence of vesicle rupture time on bulk vesicle concentration was observed. Ultimately, this work elicits fundamental insight into the importance of flow and bulk vesicle concentration in the adsorbed vesicle rupture process during SLB formation using QCM-D.