The stem cell niche represents a heterogeneous environment supporting mesenchymal stem cell (MSC) self-renewal and differentiation. However, the mechanisms with which niche signals maintain self-renewal or direct differentiation remain unclear. The objective of this study was to develop an approach to visualize and identify niche signals that maintain MSC self-renewal and differentiation. A culture system was developed where laser tweezers manipulate and encapsulate individual cells into specific positions within a three-dimensional (3D) scaffold and signaling kinetics are regulated by microfluidic devices, controlled release microspheres, or other niche cells. This system was designed to control individual cells and the signals they receive. To validate laser tweezer manipulation, we employed the U937 cell line prior to MSCs because they easily differentiate into macrophages by diffusible small molecules (phorbol 12-myrisate 13-acetate; PMA). Poly (ethyleneglycol) diacrylate (PEGDA) and alginate were analyzed as a scaffold for 3D culture systems. PEGDA is an easily modified synthetic matrix that is rapidly photopolymerizable in a controlled manner and permits signal and waste diffusion. Alginate is a natural polymer that facilitates nutrient and waste diffusion and is rapidly polymerized by Ca2+ ions. U937 cells and MSCs were successfully manipulated by laser tweezers into homotypic 4x4 arrays and encapsulated in the 3D scaffolds. This approach was also successful in forming heterotypic arrays of U937 and MSC with various cell types. Large cell arrays were formed by juxtaposing several smaller cell arrays. In order to monitor cell metabolic activity and differentiation in real-time, fluorescent reporter constructs were developed. Constituent promoter CMV-driven and differentiation induced promoters fluorescence constructs were designed to visualize cellular metabolic activity of individual cells and to monitor MSC differentiation. The PPARγ, collagen2a, and osteopontin promoters are indicative of adipose, cartilage, and bone differentiation.