Human embryonic stem cells (hESC) are self-renewing pluripotent cells capable of differentiating into cells representative of all three embryonic germ layers. Hence, they hold great potential for regenerative medicine. However, significant cell numbers are required to fulfill their potential therapeutic applications. In this study, perfusion with supplemented conditioned media (SCM), produced by mouse embryonic fibroblasts (MEF), was adopted to improve cell densities of hESC cultures. Perfusion enhanced hESC numbers by 70% compared to static conditions, on both organ culture dish (OCD) and petri dish cultures. All cultures maintained healthy expression of the pluripotent marker, Oct-4 transcription factor. In vivo, perfused hESC formed teratomas in severe combined immunodeficiency (SCID) mice models that represent the three embryonic germ layers. When SCM was produced with lower concentrations of MEF, hESC densities and Oct-4 levels were reduced. Hence, perfusion with SCM is a potential feeding method for scale-up production of hESC.