A thermoresponsive substrate based on a triblock copolymer, poly(N-isopropylacrylamide)-block-poly[(R)-3-hydroxybutyrate]-block-poly(N-isopropylacrylamide) (PNIPAAm-PHB-PNIPAAm), co-coated with gelatin, was developed for the culture and non-enzymatic recovery of mouse embryonic stem cells. After culture, the cells could be detached by cooling at 4 degrees C for 20 min without trypsin digestion. The embryonic stem cells remained undifferentiated after culture on the gelatin/copolymer-coated surfaces, similar to standard culture techniques. Overall, the triblock copolymer coating was superior to the PNIPAAm homopolymer coating in terms of supporting better cell growth, being more stable, presenting a more homogeneous surface coating, and maintaining pluripotency of the embryonic stem cells.