A large-scale stirred culture system for the expansion of mouse embryonic stem cells (mESCs) in spinner flasks under serum-free conditions was established using macroporous microcarriers for cell attachment and growth. This type of microcarrier was chosen as it potentially offers more protection to cells against shear stress in the absence of serum compared to microporous ones. In addition, methods to characterize ESCs after large-scale expansion were established. The pluripotency of expanded mESCs was evaluated based on both flow cytometry and alkaline phosphatase staining. Envisaging the application of ESCs as a potential source of neural progenitors, the neural commitment potential of cells after expansion in the spinner flask was also determined by culturing cells in serum-free adherent monolayer conditions.