A spatially organized three-dimensional (3D) co-culture of multiple cell types is required to recapitulate cellular interactions and microenvironments in complex tissues. Although there are limited reports for 3D patterning of cells and materials, approaches to examine functional interactions of 3D spatially patterned multiple cell types are lacking entirely. This is mostly due to difficulties in controlling the physical arrangement of cells in a 3D matrix and the physical properties of the cell-encapsulating matrix, while keeping the cells alive and functional for extended periods of time. In this study, an automated maskless fabrication technique is combined with a tunable polymer blend to spatially organize primary hippocampus neurons (HNs) and skeletal muscle myoblast cells (MCs) in a 3D hydrogel matrix with tunable mechanical and degradation properties. The spatial organization of these multiple cell types revealed that the presence of MCs resulted in increased cholinergic functionality of the HNs, as quantified by their choline acetyltransferase activity. The presence of a factor alone is not sufficient, but its spatiotemporal control is necessary; a condition that is possibly true for many cellular interactions. Therefore, the system described here offers a different approach to examine such previously unknown interactions. The approach proposed in this study can be used to examine interactions between many different cell types and shift the 3D fabrication paradigm to a next level, which is to fabricate tissues that are not only viable but also functional.