A 12-hour pulse of RUNX1 expression is sufficient to rescue continuous Pu.1 mRNA expression, induce Csf1r mRNA expression, and rescue subsequent hematopoietic development. ES cells were differentiated as indicated. mRNA of the indicated cell populations (iRUNX1 and wt) was measured by real-time PCR (A) and RUNX1 protein was measured by Western blotting (B) using an anti-RUNX1 antibody after 4 days of blast culture (except for the +DOXd2 samples, which were measured before or after 8 hours of withdrawal, as indicated). (C) Number of hematopoietic colonies before and after DOX withdrawal; (D) Giemsa-stained cytospin of representative colonies. mRNA of iRUNX1 Flk1⁺ cells was prepared straight after cell sorting. Numbers in panels A and C represent mean values of 2 independent experiments analyzed in duplicate.