The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells.