Better experimental models are needed to enhance our understanding of metabolic regulation which is seen in obesity and metabolic disorders, such as type 2 diabetes. In vitro models based on microfluidics enable physiological representations of tissues with several advantages over conventional culture systems, such as perfused flow to better mimic the physiological environment. Although cell lines such as 3T3-L1 have been incorporated in microfluidic devices, murine primary preadipocytes have not been differentiated and maintained for long-term monitoring in these culture systems. We describe the differentiation of these cells into white adipose depots on a perfused microfluidic chip. We compare the effects of shear flow on these cells, and show with a direct comparison of high/low shear conditions that direct shear is detrimental to the viability of preadipocytes. We further develop a dual-chamber microfluidic chip that enables perfusion while at the same time protects the cells from direct fluidic shear. We show that the dual-layer microfluidic device enables long-term culture of cells and allows stimulation of cells through perfusion-we can culture, differentiate, and maintain the differentiated adipose tissue for over multiple weeks in the device. Both triglycerides and lipolytic glycerol production increased significantly by several folds during differentiation. After successful differentiation, the adipocytes had upregulated expression of leptin and adiponectin, which are important makers of the final stage of adipogenic differentiation. In conclusion, the dual-layer microfluidic device incorporated with primary adipocytes improves the understanding of adipose differentiation under dynamic conditions and is positioned to serve as a disease model for studying obesity and other metabolic disorders.