Separation of differentiated and undifferentiated cells without labeling is required for cell analyses and clinical application of cultured differentiated cells in vitro. To proceed with the passive separation of differentiated cells inside a clean bench, we developed a system of deterministic lateral displacement (DLD) microfluidic devices and applied this system to sort differentiated cells in vitro. The fluid flow is driven by compressed air to the buffer. Priming and sorting can be completed by air pressure control. We use this system to separate C2C12 mononuclear myocytes from multinuclear myotubes. Additionally, using a DLD microfluidic channel of Dc = 20 μm, multinuclear myotubes can be effectively sorted as larger particles. We prepared differentiated adipocytes from mouse embryonic fibroblast (MEF) cells and sorted those containing lipid droplets. The diameters of these sorted adipocytes considered larger particles, exceeded 20 μm, similar to the Dc of the DLD microfluidic channel. Differentiated cell sorting by cell size will contribute to single-cell analyses and in vitro tissue model preparation for drug discovery.