Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self-renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development. Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self-renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development.