Although previous studies suggest that proplatelet formation in megakaryocytes involves caspase-3, the mechanism underlying the activation of caspase-3 is unknown. Here, we analyzed caspase activation in a human megakaryoblastic cell line, MEG-01, which forms proplatelets spontaneously. Specific activation of caspase-3 and caspase-4 was found in proplatelets. Consistent with previous observations of caspase-4 autoactivation in response to endoplasmic reticulum (ER) stress, several ER stress marker proteins were expressed during proplatelet formation. A pharmacological ER stressor enhanced platelet production via proplatelet formation, whereas inhibition of caspase-4 caused suppression. These results suggest that ER stress is a mechanism underlying the maturation of megakaryocytes.