Fetal and adult Leydig cells develop in mammalian prenatal and postnatal testes, respectively. In mice, fetal Leydig cells (FLCs) emerge in the interstitial space of the testis at embryonic day 12.5 and thereafter increase in number, possibly through differentiation from progenitor cells. However, the progenitor cells have not yet been identified. Previously, we established transgenic mice in which FLCs are labeled strongly with enhanced green fluorescent protein (EGFP). Interestingly, fluorescence-activated cell sorting provided us with weakly EGFP-labeled cells as well as strongly EGFP-labeled FLCs. In vitro reconstruction of fetal testes demonstrated that weakly EGFP-labeled cells contain FLC progenitors. Transcriptome from the 2 cell populations revealed, as expected, marked differences in the expression of genes required for growth factor/receptor signaling and steroidogenesis. In addition, genes for energy metabolisms such as glycolytic pathways and the citrate cycle were activated in strongly EGFP-labeled cells, suggesting that metabolism is activated during FLC differentiation.