Cell therapy using embryonic stem cells (ESCs) requires reproducible, well-defined culture systems for producing large numbers of well-characterized cells. Current ESC differentiation process involving three steps, expansion of ESCs, formation of embryoid bodies and induction of lineage-specific differentiation, is laborious and difficult to scale-up. In this study, a simple process using fibrous bed bioreactors was developed for integrated ESC expansion and differentiation. When cultured in a PET fibrous matrix with a growth medium for 30 days, ESCs were expanded 125-fold to reach a high cell density of 1×10^8 cells/ml matrix without significant differentiation. Following the expansion of ESCs in a fibrous bed bioreactor, continually refreshing the bioreactor with a differentiation medium induced ESC differentiation. Multi-lineage differentiation was confirmed using immunocytochemistry and FACS analyses. This simplified, integrated ESC expansion and differentiation process can meet the stringent requirements for developing large-scale cultures for producing ESCs for potential clinical applications.