Pluripotent stem cells are under the influence of soluble factors in a diffusion dominant in vivo microenvironment. In order to investigate the effects of secreted soluble factors on embryonic stem cell (ESC) behavior in a diffusion dominant microenvironment, we cultured mouse ESCs (mESCs) in a membrane-based two-chambered micro-bioreactor (MB). To avoid disturbing the cellular environment in the top chamber of the MB, only the culture medium of the bottom chamber was exchanged. Cell growth in the MB after 5 days of culture was similar to that in conventional 6-well plate (6-WP) and membrane-based Transwell insert (TW) cultures, indicating adequate nutrient supply in the MB. However, the cells retained higher expression of pluripotency markers (Oct4, Sox2 and Rex1) and secreted soluble factors (FGF4 and BMP4) in the MB. Inhibition of FGF4 activity in the MB and TW resulted in a similar cellular response. However, in contrast to the TW, inhibition of BMP4 activity revealed that autocrine action of the upregulated BMP4, which acted cooperatively with leukemia inhibitory factor (LIF), upregulated the pluripotency markers expression in the MB culture. We propose that BMP4 accumulated in the diffusion dominant microenvironment of the MB upregulated its own expression by a positive feedback mechanism—in contrast to the macro-scale culture systems—thereby enhancing the pluripotency of mESCs. The micro-scale culture platform developed in this study enables the investigation of the effects of soluble factors on ESCs in a diffusion dominant microenvironment, and is expected to be used to modulate the ESC fate choices.