Cellular microenvironment consists of intercellular contacts, secreted factors and extracellular matrix which define and dynamically modulate cell function and phenotype. The goal of this project was to increase complexity of the in vitro cellular microenvironment by assembling three distinct cell phenotypes into microfabricated tri-cultures. Surface micropatterning combined with sequential cell seeding enabled the placement of three cell types on the same surface. Our surface micropatterning approach involved photolithographic patterning of collagen (I) to create cell-adhesive domains on glass surfaces modified with acrylated silane. The photosensitive poly(ethylene glycol) PEG prepolymer was then micropatterned in registration with the collagen (I) domains. The PEG hydrogel photolithography was central to the development of triple cell cultures. This top-down approach developed by us earlier offers the flexibility of traditional resist lithography in permitting to align and juxtapose several microfabricated layers. The merger of photolithographic protein patterning with PEG hydrogel photolithography allowed to modulate biointerfacial properties from cell-adhesive collagen (I) domains to moderately non-adhesive silane regions to non-fouling PEG hydrogel microdomains. Importantly, protocols for modification of the glass substrates with the acrylated silane molecules were optimized to result in delamination of the PEG gel microstructures after ~48 hrs under cell culture conditions. Triple cell cultures were assembled using a succession of seeding steps built around the deprotection of PEG gel covered regions of the glass substrate: 1) seeding of hepatocytes resulted in cell attachment to collagen regions only, 2) fibroblast (or stellate cell) seeding resulted in cell attachment to silane regions and creation of micropatterned co-cultures, 3) upon delamination of PEG gel elements, transformed fetal hepatocytes were seeded on to the surface leading to the formation of microfabricated triple cell cultures. In conclusion, we developed a surface engineering approach incorporating both traditional photoresist lithography and a novel biomaterial microfabrication method. The microfabricated triple cell cultures may provide an “instructive” hepatic microenvironment required for the induction of mature hepatic function in fetal hepatocytes or stem cells.