Transient Smad1 expression also expands macrophage, megakaryocyte, and definitive mixed-lineage progenitors. Developing iSmad1 EBs were left untreated (no dox) or treated with doxycycline according to the day 2 washout protocol. EBs were then harvested at day 5.75 and replated to score for (A) macrophage progenitors (MacPs), (B) megakaryocyte progenitors (MegaPs), or (C) mixed-lineage progenitors (Mix). After plating, the macrophage and megakaryocyte colonies were scored on day 7, and the mixed-lineage colonies were scored on day 8. In panels A and B, the doxycycline was added for 6 hours at day 2, and a subset of EBs was also exposed to doxycycline at the time of replating in methylcellulose (mec dox) to test if this inhibits differentiation, which it does not. In panel C, the iSmad1 EBs were untreated (no dox) or treated with doxycycline for 6 hours on day 2 (d2 wo), day 3 (d3 wo), or day 4 (d4 wo). Induction at later time points did not expand the number of mixed-lineage colonies (not shown). In all cases, for each sample, n = 3. Error bars indicate the SEM and the asterisks indicate a P < .01 compared to the no dox samples.