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Surface biomaterials and topography of extracellular matrices (ECMs) is critical for the culture of various cells and modern tissue engineering. However, the potential of ECMs are relatively not much focused as an important factor in those purposes. Actually, the very first cell differentiation at the transition from an eight-cell embryo to a blastocyst occurs within an ECM called the zona pellucida during early mammalian embryonic development. Therefore the zona pellucida can be considered to be one of the best candidate ECMs for cell cultures, if it could be clearly separated. In addition, the natural three dimensional spherical structure of the zona pellucida would also be effective for embryoid body (EB) formation. However, to obtain the zona pellucida keeping its original spherical form without splitting of the membrane is quite difficult due to its small size (<100 µm in diameter). Here, we extruded the yolk and remaining inner mass from fertilized zebrafish eggs and used the resulting chorions as an extracellular matrix for the differentiation and attachment of mouse P19 embryonic carcinoma (EC) cells. Generally the chorion of the carp like zebrafish is transparent and contains many penetrating nanopores, (500≈700 µm in diameter). Moreover, the membrane protein of zebrafish chorion (ZP2) is known to exhibit a similar degree of homology with the mouse zona pellucida proteins ZP1 and ZP2. The chorion is also relatively easy to be collected because zebrafish eggs require just 2 days for developing from embryos to fry. Cells inserted into the chorion showed the spontaneous formation of embryoid body due to the repulsive cell adhesion of the chorion and differentiated specifically into neural cells and cardiomyocytes. These results indicate that chorions from fertilized zebrafish eggs may be used as an extracellular matrix alternative and applied for cellular differentiation to specific cell lineages. [ABSTRACT FROM AUTHOR]