The spatial organization of cells of different phenotypes is an important and often defining determinant of tissue function. In tissue engineering, which attempts to rebuild functional tissues from cellular and synthetic components, spatial patterning of cells onto biomaterials is likely to be equally important. We have printed combinatorial arrays of extracellular matrix (ECM) and screened them for attachment by HepG2 hepatocytes, LX-2 hepatic stellate cells, primary portal fibroblasts, and bovine aortic endothelial cells-cells selected as representative phenotypes found in adult liver. Differential cell attachment to the underlying matrix proteins allowed us to establish two-dimensional co-cultures of HepG2 with these non-parenchymal cell types. These general approaches were then translated to tissue engineering scaffolds where deposition of ECM proteins onto electrospun polylactide meshes resulted in patterned HepG2 cultures. We observed that the spatial organization of fibronectin deposits influenced HepG2 attachment and the establishment of co-cultures on our arrays. These micropatterned co-culture systems should serve as valuable tools for studying the soluble and insoluble signals involved in liver development, function, and disease.