Pluripotency of stem cells refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. Maintaining pluripotent stem cells in culture is a tedious and demanding task. Monitoring the changing pluripotency in live cells is essential for this task. Here, we report a pluripotency monitoring system in which the expression of green fluorescent protein (GFP) is under the control of the promoter of a pluripotency gene (Rex-1). The reporter system can be permanently integrated into the genome of live cells via lentiviral vectors. This pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single cell and its progeny. Our data demonstrate that the BJ cell line (a normal human fibroblast cell line) that carries this hRex-GFP construct does not express GFP until it is reprogrammed to pluripotent stage. The GFP expression was progressively lost when these pluripotent hRex-GFP cells exposed to differentiation conditions. These results indicate that insertion of the hRex-GFP construct is stable in descendant cells, a finding that has particular value in tracking pluripotency of transplanted cells and their progenies in animal studies. With this hRex-GFP reporter, the pluripotency of cells can be monitored over long periods of time via the expression of GFP. Use of this reporter system will facilitate the study of stem cell pluripotency at the single-cell level, and sheds light on the molecular mechanisms of stem cell self-renewal and subsequent differentiation.