Exogenous cues involved in the regulation of the initials steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse ES (embryonic stem) cell differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that Bone Morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ES cell-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations, preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell replacement strategies.