In native tissues, cells reside in a complex microenvironment (niche) consisting of factors including neighbor cells, soluble factors, extracellular matrices, topological and mechanical signals. Cell niche is critical in maintaining their phenotype and determining their fates and functions. Reconstituting complex cell niche factors in vitro, either individually or in combinations, in a quantitatively and spatially controllable manner, is critical for investigating the interactions between cells and their niches and hence deriving designing strategies for optimal conditions during cell culture applications and optimal scaffolds for tissue engineering applications. Our lab has developed a multiphoton microfabrication and micropatterning (MMM) technology. Here, the technical capability of the MMM platform in fabricating complex protein microstructures and micropatterns with pre-designed topological features, mechanical properties, extracellular matrix, cell interaction molecules and soluble factors, and biomedical applications including cell niche factor screening for phenotype maintenance and engineering cell niche for cell fate determination will be discussed.