Adipogenesis and increase in fat tissue mass are mechanosensitive processes and hence should be influenced by the mechanical properties of adipocytes. We evaluated subcellular effective stiffnesses of adipocytes using atomic force microscopy (AFM) and interferometric phase microscopy (IPM), and we verified the empirical results using finite element (FE) simulations. In the AFM studies, we found that the mean ratio of stiffnesses of the lipid droplets (LDs) over the nucleus was 0.83 ± 0.14, from which we further evaluated the ratios of LDs over cytoplasm stiffness, as being in the range of 2.5 to 8.3. These stiffness ratios, indicating that LDs are stiffer than cytoplasm, were verified by means of FE modeling, which simulated the AFM experiments, and provided good agreement between empirical and model-predicted structural behavior. In the IPM studies, we found that LDs mechanically distort their intracellular environment, which again indicated that LDs are mechanically stiffer than the surrounding cytoplasm. Combining these empirical and simulation data together, we provide in this study evidence that adipocytes stiffen with differentiation as a result of accumulation of LDs. Our results are relevant to research of adipose-related diseases, particularly overweight and obesity, from a mechanobiology and cellular mechanics perspectives.