The objective of this research was to determine the appropriate cell culture conditions for embryonic stem (ES) cell proliferation and differentiation in fibrin scaffolds by examining cell seeding density, location, and the optimal concentrations of fibrinogen, thrombin, and aprotinin (protease inhibitor). Mouse ES cells were induced to become neural progenitors by adding retinoic acid for 4 days to embryoid body (EB) cultures. For dissociated EBs, the optimal cell seeding density and location was determined to be 250,000 cells/cm(2) seeded on top of fibrin scaffolds. For intact EBs, three-dimensional (3D) cultures with one EB per 400 microL fibrin scaffold resulted in greater cell proliferation and differentiation than two-dimensional (2D) cultures. Optimal concentrations for scaffold polymerization were 10mg/mL of fibrinogen and 2 NIH units/mL of thrombin. The optimal aprotinin concentration was determined to be 50 microg/mL for dissociated EBs (2D) and 5 microg/mL for intact EBs in 3D fibrin scaffolds. Additionally, after 14 days in 3D culture EBs differentiated into neurons and astrocytes as indicated by immunohistochemisty. These conditions provide an optimal fibrin scaffold for evaluating ES cell differentiation and proliferation in culture, and for use as a platform for neural tissue engineering applications, such as the treatment for spinal cord injury.