Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number <1), cells are affected by apparent nutrient depletion and waste accumulation, evidenced by reduced cell expansion and altered morphology. At higher rates, cells are spontaneously washed out, and display morphological changes which may be indicative of early-stage differentiation. However, between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system, with regular morphology and maintenance of the pluripotency marker TG30 in >95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture. Biotechnol. Bioeng. 2011;108: 2894–2904. © 2011 Crown in the right of Canada.