This study explores the role of the transcription factor RUNX3 in early blood cell development using an ex vivo embryonic stem cell (ESC) differentiation system. ESCs were cultured to form embryoid bodies and further differentiated in the presence or absence of doxycycline to induce RUNX3 expression. Flow cytometry was used to assess the expression of key hematopoietic markers, including CD41, CD45, and c-Kit. The results showed that RUNX3 induction increased the proportion of c-Kit/CD41 double-positive early blood progenitors. Additionally, real-time PCR analysis confirmed that myb, a gene essential for hematopoietic progenitor development, was upregulated in RUNX3-induced cells, further supporting RUNX3’s role in promoting hematopoietic precursor formation during early development.