Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian pre-granulosa cells or testicular Sertoli cells. This fate specification hinges on a balance of transcriptional control. Here we report that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes pre-granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity and combined loss of RUNX1 and FOXL2 results in masculinization of fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target common sets of genes. These findings identify RUNX1, with an ovary-biased expression pattern conserved across species, as a regulator in securing the identity of ovarian-supporting cells and the ovary.