The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn
-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0–40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture.