Tethered bilayer lipid membranes (tBLMs) are designed on mixed self-assembled monolayers (SAMs) of a novel synthetic anchoring thiol, 2,3-di-o-palmitoylglycerol-1-tetraethylene glycol mercaptopropanoic acid ester (TEG-DP), and a new short dilution thiol molecule, tetraethylene glycol mercaptopropanoic acid ester (TEG). tBLM formation was accomplished by self-directed fusion of small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The influence of the dilution of the anchoring thiol molecule in the SAM on the vesicle fusion process and on the properties of the resulting tBLMs is studied. It is observed by quartz crystal microbalance that vesicle fusion is a one-step process for a pure TEG-DP SAM as well as for mixed SAMs containing a high concentration of the anchoring thiol. However, upon dilution of the anchoring thiol to moderate concentrations, this process is decelerated and possibly follows a pathway different from that observed on a pure TEG-DP SAM. Electrochemical impedance spectroscopy is used to qualitatively correlate the composition of the SAM to the electrical properties of the tBLM. In this paper we also delineate the necessity of a critical concentration of this anchoring TEG-DP thiol as a requisite for inducing the fusion of vesicles to form a tBLM.