ESCs have unlimited proliferation potential and capability to differentiate into all tissue types. They are ideal cell sources for tissue engineering and cell therapy, but their supplies are limited. Current in vitro ESC cultures are carried out in tissue flasks with the surface precoated with extracellular matrix (ECM) proteins. T-flask cultures also require frequent subculturing because their limited surface area cannot support long-term growth of ESCs. In this work, ECM coating and frequent subculturing required in two-dimensional (2D) cultures were circumvented by culturing murine ESCs in three-dimensional (3D) polyethylene terephthalate (PET) fibrous matrices. Also, media conditioned with STO fibroblast cells were used to replace leukemia inhibitory factor and to effectively maintain the pluripotency of murine ESCs in a long-term static culture. However, the lactic acid present in the conditioned medium could inhibit ESC growth and induce spontaneous differentiation when its concentration exceeded 1.5 g/l. In addition, the 3D static culture could be limited by oxygen, which was depleted in the long-term culture when cell density in the matrix was high. However, these problems can be alleviated in dynamic culture with improved oxygen transfer and continuous media perfusion. The matrix pore size also had profound effects on ESCs. The smaller-pore (30-60 mum) matrix gave a higher proliferation rate and Oct-4 and stage specific embryonic antigen-1 expressions. Overall, the 3D culturing method is superior to the 2D culture method and can provide an economical way to mass-produce undifferentiated ESCs in uncoated matrices and conditioned media.