The various cell mechanisms, including cell-cell interactions, in native tissue could be better understood by engineering a cell coculture with a micro environment that closely mimics the natural cell arrangement. To this end, we developed a cell micropatterning system that uses a weakly focused laser beam to trap individual cells at the center of the beam and propel them forward onto an appropriate substrate. The optimal methods of introducing different cell types to be patterned into the patterning system and preventing cells from randomly falling onto the pattern were issues to be addressed with this system. Here, we report the development of a multi-chamber, multi-beam laser cell micropatterning system, in which the delivery of specific cells into the beam can be controlled using secondary laser beams. This permits consecutive creation of a pattern involving multiple cell types at specific relative positions. As examples, various patterns of fibroblasts have been created on collagen coated coverslips. In addition, two asynchronously beating clusters of cardiomyocytes were connected with fibroblasts of cardiac origin, yielding a deeper insight into the electrophysiological role of fibroblasts in conduction of the action potentials among cardiomyocytes.