The effects of pore size in a 3‐D polyethylene terephthalate (PET) nonwoven fibrous matrix on long‐term tissue development of human trophoblast ED₂₇ cells were studied. Thermal compression was used to modify the porosity and pore size of the PET matrix. The pore size distributions in PET matrices were quantified using a liquid extrusion method. Cell metabolic activities, estradiol production, and cell proliferation and differentiation were studied for ED₂₇ cells cultured in the thermally compressed PET matrices with known pore structure characteristics. In general, metabolic activities and proliferation rate were higher initially for cultures grown in the low‐porosity (LP) PET matrix (porosity of 0.849, average pore size of 30 μm in diameter) than those in the high‐porosity (HP) matrix (porosity of 0.896, average pore size of 39 μm in diameter). However, 17β‐estradiol production and cell differentiation activity in the HP matrix surpassed those in the LP matrix after 12 days. The expression levels of cyclin B1 and p27^kip1 in cells revealed progressively decreasing proliferation and increasing differentiation activities for cells grown in PET matrices. Also, difference in pore size controlled the cell spatial organization in the PET matrices and contributed to the tissue development in varying degrees of proliferation and differentiation. It was also found that cells grown on the 2‐D surface behaved differently in cell cycle progression and did not show increased differentiation activities after growth had stopped and proliferation activities had lowered to a minimal level. The results from this study suggest that the 3‐D cell organization guided by the tissue scaffold is important to tissue formation in vitro. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 606–618, 2000.