Obesity is associated with several comorbidities that cause high mortality rates worldwide. Thus, the study of adipose tissue (AT) has become a target of high interest because of its crucial contribution to many metabolic diseases and metabolizing potential. However, many AT-related physiological, pathophysiological, and toxicological mechanisms in humans are still poorly understood, mainly due to the use of non-human animal models. Organ-on-chip (OoC) platform is a promising alternative to animal models. However, the use of adipose-derived human mesenchymal stem cells (hASCs) in these models is still scarce, and more knowledge on the effects properties of culturing hASCs in OoC models is needed. Here, we present the development of an OoC using hASCs to assess adipogenic differentiation. The device capability to increase hASC differentiation levels was confirmed by Nile red staining to verify lipid droplets inside cells after 10 days of culture and fluid flow of 10 µL/h. The Adipo-on-a-chip system increases hASC proliferation and differentiation area compared with the standard culture method under static conditions (96-well plates) verified in hASCs from different donors by image analysis of cells stained with Nile red. The expression of the gene FABP4 is lower in the MPS, which calls attention to different homeostasis and control of lipids in cells in the MPS, compared with the plates. An increase of hASC proliferation in the MPS related to the 96-well plate was verified through protein Ki-67 expression. Cell and nuclei morphology (area, roundness, perimeter, width, length, width to length rate, symmetry, compactness, axial and radial properties to nuclei, and texture) and dominant direction of cells inside the MPS were evaluated to characterize hASCs in the present model. The presented microphysiological system (MPS) provides a promising tool for applications in mechanistic research aiming to investigate adipogenesis in AT and toxicological assessment based on the hASC differentiation potential.