RUNX1 is essential for E2A-PBX1–mediated HSPC transformation in vitro. DNA-binding function of E2A-PBX1 is dispensable in HSPC transformation. Murine HPSCs transduced with control or indicated E2A-PBX1–expressing retroviruses were subjected to assays for (A) proliferation kinetics in a liquid culture and (B) in vitro serial replating to score CFUs in a semisolid culture. RUNX1 depletion significantly impairs the maintenance of E2A-PBX1-transformed HSPCs. (C) Cell proliferation and (D) CFU assays of E2A-PBX1–transformed HSPCs transduced with lentiviruses expressing Scramble (shControl) or RUNX1 (sh574 and sh812) shRNAs. Statistics by Student t test. **P < .01. NS, not significant.