Supplementary Figure 1. Current definitive endoderm (DE) differentiation protocols are neither efficient nor robust. A previously published DE protocol (8) applied to several hESC lines (H1, H9, HUES1, HUES9) results in poor DE induction (n=3 biologically independent experiments; (mean± S.D.). Supplementary Figure 2. Cell line specific effects of extracellular matrix proteins (ECMPs) in promoting differentiation of hESCs to definitive endoderm (DE). (a) Dendrogram of similarity relationship between biologically independent array experiments demonstrates the consistent effects of the ECMP combinations within each hESC line. (b) The Pearson correlation coefficient matrix reveals the hESC linespecific effects of certain ECMP in promoting efficient DE differentiation. Supplementary Figure 3. Definitive endoderm induction on extracellular matrix protein (ECMPs) in additional hESC lines. Representative images of anti-SOX17 immunofluorescence of (a) HUES1 hESCs and (b) H9 hESCs differentiated to definitive endoderm (DE) on Fibronectin (FN), Vitronectin (VTN), and Fibronectin and Vitronectin (FN + VTN) (mean± S.E.M.). Gene expression analysis of endoderm genes SOX17 and FOXA2 of (c) HUES1 hESCs and (d) H9 hESCs differentiated to definitive endoderm (DE) on Fibronectin (FN), Vitronectin (VTN), and Fibronectin and Vitronectin (FN + VTN) (mean± S.E.M.). Supplementary Figure 4. Expression of laminin (LN) integrin subunits in definitive endoderm (DE). (a) Time course of laminin specific subunits ITGA6 (CD49f) during hESC (H1, H9, HUES1) to DE (n=3). (b) Representative flow cytometry histograms of cell surface protein expression of laminin specific subunits ITGA6 (CD49f) and ITGB1 (CD29) in hESCs and DE. (c) Quantification of percentage of cell surface protein expression of ITGA6+ (CD49f+) and ITGB1+ (CD29+) hESCs and DE (HUES9 and HUES1; n=3; error bars, S.E.M.). Supplementary Figure 5. RFP and integrin expression in doxcycline (DOX) treated hESCs. (a) RFP expression marks inducible shRNA expression (mean ± S.E.M.). (b-c) qPCR analysis of DOX treated ITGA5shRNA and ITGAVshRNA hESCs. Analysis reveals that ITGAV (CD51) and ITGA5 (CD49e) gene expression is unchanged in DOX treated ITGA5shRNA and ITGAVshRNA hESCs, respectively. (d) Wild-type HUES9 hESCs were treated with DOX for 72 hrs. Quantitative PCR (qPCR) analysis shows DOX treatment alone does not affect ITGA5 (CD49e) and ITGAV (CD51) gene expression. (n=3; error bars, S.E.M). Supplementary Figure 6. Quantitative PCR (QPCR) analysis of hESC derived definitive endoderm (DE) sorted for integrin α5 (ITGA5/CD49e) and integrin αv (ITGAV/CD51). Gene expression analysis of the sorted populations reveals that double positive cells [ITGA5(CD49e)+ / ITGAV(CD51)+] expressed significantly higher levels of SOX17 than single positive cells [ITGA5(CD49e)+ / ITGAV(CD51)- or ITGA5(CD49e)- / ITGAV(CD51)+] or double negative cells (ITGA5(CD49e)- / ITGAV(CD51)-) (n=3; error bars, S.E.M). Supplementary Table 1. Raw data of heatmap generated in Figure 1d. Supplementary Table 2. Antibodies used in this study. Supplementary Table 3. Taqman gene expression assay primers used in this study.