AbstractWe found by electron microscopy that the inter‐membrane space of embryonic stem cells is irregular and generally wider than in differentiated cells. Among a panel of nuclear envelope structural proteins examined, the expression of Syne1/nesprin‐1 was found to be greatly induced upon differentiation. Down‐regulation of Syne1 by siRNA in differentiated embryonic stem cells caused the nuclear envelope to adopt a configuration resembling that found in undifferentiated embryonic stem cells. Suppression of Syne1 expression did not produce a detectable impact on the retinoic acid–induced differentiation of embryonic stem cells; however, forced expression of Syne1 enhanced the tendency of the cells to lose pluripotency. Thus, we found that low expression of Syne1 splicing isoforms accounts for the wider and irregular nuclear envelope inter‐membrane space in embryonic stem cells. We conclude that the nuclear envelope structural change accompanying differentiation likely participates in promoting the differential chromatin organization of the differentiated cells. Developmental Dynamics 240:2245–2255, 2011. © 2011 Wiley‐Liss, Inc.