Differentiation methods often rely exclusively on growth factors to direct mouse embryonic stem cell (ESC) fate, but the niche also contains fibrillar extracellular matrix (ECM) proteins, including fibronectin (FN) and laminin, which could also direct cell fate. Soluble differentiation factors are known to increase ECM expression, yet ECM's ability to direct ESC fate is not well understood. To address the extent to which these proteins regulate differentiation when assembled into a matrix, we examined mouse ESC embryoid bodies (EBs) and found that their ability to maintain pluripotency marker expression was impaired by soluble serum FN. EBs also showed a spatiotemporal correlation between expression of FN and GATA4, a marker of definitive endoderm (DE), and an inverse correlation between FN and Nanog, a pluripotency marker. Maintenance of mouse ESC pluripotency prevented fibrillar matrix production, but induction medium created lineage-specific ECM containing varying amounts of FN and laminin. Mouse ESC-derived matrix was unlike conventional fibroblast-derived matrix, which did not contain laminin. Naïve mouse ESCs plated onto ESC- and fibroblast-derived matrix exhibited composition-specific differentiation. With exogenously added laminin, fibroblast-derived matrix is more similar in composition to mouse ESC-derived matrix and lacks residual growth factors that mouse ESC matrix may contain. Naïve mouse ESCs in DE induction medium exhibited dose-dependent DE differentiation as a function of the amount of exogenous laminin in the matrix in an α3 integrin-dependent mechanism. These data imply that fibrillar FN is necessary for loss of pluripotency and that laminin within a FN matrix improves DE differentiation.