We present a simple method to pattern multiple cell populations inside a microfluidic channel. The microchannel is partially filled with a cell suspension, and the position of the liquid boundary remains pinned by surface tension. Cells then adhere only in the filled portion of the channel, producing a very sharp boundary. The process can be performed in an unmodified microfluidic channel with only a manual syringe and can be repeated multiple times to pattern cocultures or tricultures. We demonstrate the patterning method with two different mammalian cell types, 3T3 fibroblasts and NMuMG epithelial cells, and channel heights of 1.5 mm and 0.5 mm. We anticipate that this method will be useful for studies of cell-cell interactions where precise control of the fluidic microenvironment is required.