Nuclear lamins comprise the nuclear lamina, a scaffold-like structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription–polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.