The stiffness of nanoscale liposomes, as measured by atomic force microscopy (AFM), was investigated as a function of temperature, immobilization on solid substrates, and cantilever tip shape. The liposomes were composed of saturated lipids and cholesterol, and the stiffness values did not change over the temperature range of 25-37 °C and were independent of immobilization methods. However, the stiffness varied with the tip shape of the cantilever. Therefore, 24 cantilevers were evaluated in terms of tip shape and aspect ratio (length/width) via a nonblind tip reconstruction (NBTR) method that used a tip characterizer with isolated line structures having specified dimensions. A standard for screening the tip geometry was established. A 24-fold improvement in stiffness precision in terms of relative standard deviation was demonstrated by using at least three cantilevers that meet the criteria of having a tip aspect ratio greater than 2.5 and a quadratic tip shape function. A significant difference in stiffness was subsequently revealed between dipalmitoylphosphatidylcholine-cholesterol (1:1 molar ratio) and egg yolk phosphatidylcholine-cholesterol (1:1 molar ratio) liposomes. Tip analysis using NBTR improved the precision of AFM stiffness measurements, which will enable the control of mechanical properties of nanoscale liposomes for various applications.