Advanced approaches to direct the differentiation of embryonic stem cells are highly sought after. The surface-bound chemical gradient format is a powerful screening approach that can be deployed to study changes in stem cell behavior as a function of subtle changes in surface chemistry. Here, we investigate the spontaneous differentiation of cells derived from differentiating mouse embryoid body (mEB) cells into endoderm, mesoderm, and ectoderm following culture on surface-bound gradients of chemical functional groups in the absence of differentiation-biasing bioactive factors. Gradients were created using a diffusion-controlled plasma polymerization technique. The generated coating ranged from hydrophobic 1,7-octadiene (OD) plasma polymer at one end of the gradient to a more hydrophilic allylamine (AA) plasma polymer on the opposite end. The gradient surface was divided into seven equal regions of progressively increasing AA plasma polymer content and mEB cell response within these regions was compared. Cells adhered preferentially to the central regions of the gradient; however, cell proliferation increased toward AA-plasma-polymer-rich end of the gradient. Variation in the expression of germ layer markers was noted across the gradient surface. High AA:OD plasma polymer ratios triggered cell differentiation toward both mesoderm and ectoderm. Expression of tissue-specific markers, in particular, KRT18, AFP, and TNNT2, was strikingly responsive to subtle changes in surface chemistry, exhibiting vastly different expression levels between adjacent regions. Our results suggest that the surface-bound gradient platform is well suited to screening surface chemistries for use in the field of stem cell technologies and regenerative medicine.