Local micro-environmental cues consisting of soluble cytokines, extra-cellular matrix (ECM), and cell-cell contacts are determining factors in stem cell fate. These extrinsic cues form a 'niche' that governs a stem cell's decision to either self-renew or differentiate into one or more cell types. Recently, it has been shown that micro-patterning stem cells in two- and three-dimensions can provide direct control over several parameters of the local micro-environment, including colony size, distance between colonies, ECM substrate, and homotypic or heterotypic cell-cell contact. The protocol described here uses micro-contact printing to pattern ECM onto tissue culture substrates. Cells are seeded onto the patterned substrates in serum-free media and are confined to the patterned features. After patterning, stem cell phenotype is analyzed using quantitative immunocytochemistry and immunohistochemistry.