A murine embryonic stem cell (ESC) line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the smooth-muscle-specific SM22α promoter to further characterize development of the vascular smooth muscle cells (VSMCs) differentiated from ESCs is established. In SM22α-EGFP expressing ESC-derived embryoid bodies(EBs), a distinct sublineage of VSMCs could be identified by EGFP fluorescence. The SM22α promoter was switched on at day 11, and EGFP-positive cells increased gradually and reached peak at day 30. The specificity of EGFP positive cells was corroborated by RT-PCR analysis and immunostaining with antibodies against known markers for VSMCs. VSMCs were heterogeneous in their morphology in plating EBs,and could be divided into two categories: spindle-shaped or epithelioid, polygonal cells. These results suggest that SM22α-EGFP expression enables the identification of ESC -derived VSMCs by their fluorescence and morphology.