Embryonic stem cells represent considerable potential as a source of tissue-specific cells for regenerative medicine and tissue engineering. However, realizing this potential requires effective methods for guiding lineage specific differentiation of embryonic stem cells. In this work, mouse embryonic stem cells were co-cultured with adult hepatocytes to guide liver-specific differentiation of stem cells. Robotic microarraying was used to print side-by-side columns of collagen (I) and fibronectin spots on silane-modified glass substrates. When incubated with these substrates, murine embryonic stem cells (mESC) showed preference for fibronectin regions and did not appreciable attach on collagen (I) or silane domains. Addition of model hepatocytes (HepG2 cells) resulted in localization of the cells on collagen (I) domains but not on silane regions of the glass substrate. Therefore, sequential assembly of the two cell types allowed us to juxtapose clusters of hepatic cells and mESC (300 um diameter for both cell types) next to each other with ~100 um edge-to-edge spacing. The co-culture was maintained up to 2 weeks in differentiation media supplemented with insulin and dexamethasone. In order to examine the differentiation, mESC were selectively extracted from the surface using laser microdissection technique and analyzed with real-time RT-PCR. Relative expression level of each gene to housekeeping gene, b-actin, was analyzed. The expression level of marker genes for pluripotency (Oct-4 and nanog), three germ layers (alpha fetoprotein, ACTC, and tubulin), and mature hepatocytes (albumin, transthyretine, and glucose-6-phosphatase) were analyzed throughout the culture period. We observed decrease in expression pluripotency genes while endodermal genes were upregulated. Importantly, mESC cultured with HepG2 cells showed higher expression of liver-specific genes compared to mono-culture. Transthyretin expression increased up to 28.3% of that in model mouse hepatoma cell line (Hepa 1-6) after 12 days of co-culture. Co-culture also lead to significant increases in albumin expression. Albumin was increased by 13.8% compared with Hepa 1-6 while that in mono-culture was only increased by 0.9%. In conclusion, we constructed micropatterned co-cultures of mESC and adult hepatocytes on ECM microarrays. This co-culture format allowed to significantly enhance liver-specific gene expression in mESC likely through the “instructive” and “educational” effect of hepatic cells.