NOTCH and p38 function in parallel pathways to balance primitive myelopoiesis. (A) p38MAPK activation was examined in day 5 EBs by western blotting experiments under conditions of LDN50 treatment alone or in combination with 5 μM DAPT treatment, with β-actin as loading control. (B) Phospho-p38 levels were analyzed in comparison with total p38, and reported as mean ± SEM from 3 separate experiments. *P < .05 and **P < .01, respectively. Although DAPT alone decreases pp38 levels, it is not as significant as LDN, and does not impact myeloid output in the absence of LDN. (C) qPCR analysis of Dll1 and Dll3 Notch ligand genes after p38 inhibition with SCIO469. (D) Model diagram of BMP signaling control of myeloid potential proposing parallel pathways that function downstream through NOTCH and p38MAPK and converge to balance C/EBP levels. (E) The schematic summarizes ways that BMP signaling modulates primitive hematopoiesis through both SMAD-dependent and SMAD-independent mechanisms. Based on ESC differentiation studies, SMAD1 normally functions in hematovascular precursors to generally restrict myeloid and erythroid (panhematopoietic) potential. SMAD5 normally balances this effect while at the same time uniquely promoting primitive erythroid potential. BMP receptor activation separately functions to restrict C/EBPα-regulated myeloid capacity, independent of SMAD activity, through p38MAPK activation and Notch pathway inhibition.