The mechanical properties of polystyrene-block-poly(acrylic acid) (PS-b-PAA) vesicles prepared with different block copolymer chain lengths and block length ratios were studied under ambient conditions using atomic force microscopy (AFM). The deformation δ, as well as the spring constant of the membranes kmem, of individual vesicles with external diameter of ∼150 nm and systematically varied membrane thicknesses between 22 nm and 40 nm were calculated from the captured AFM force-displacement data. The application of the shell deformation model provided estimates of the apparent Young's moduli E of the vesicle membranes. While the values of kmem increased with increasing membrane thickness, the values of E were found to decrease. This observed decrease in E with increasing shell thickness coincides with the reduced degree of chain stretching reported in the literature for longer polymer chains.