Cytokines are important in adult hematopoiesis, yet their function in embryonic hematopoiesis has been largely unexplored. During development, hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, yolk sac (YS), and placenta and require the Runx1 transcription factor for their normal generation. Since IL-3 is a Runx1 target and this cytokine acts on adult hematopoietic cells, we examined whether IL-3 affects HSCs in the mouse embryo. Using Runx1 haploinsufficient mice, we show that IL-3 amplifies HSCs from E11 AGM, YS, and placenta. Moreover, we show that IL-3 mutant embryos are deficient in HSCs and that IL-3 reveals the presence of HSCs in the AGM and YS prior to the stage at which HSCs are normally detected. Thus, our studies support an unexpected role for IL-3 during development and strongly suggest that IL-3 functions as a proliferation and/or survival factor for the earliest HSCs in the embryo.