Although lymphedema was first described more than a century ago, little progress has been made in understanding the mechanisms that cause it. Investigation of the normal development of the lymphatic system has been hindered by the lack of known lymphatic-specific markers. In 1902, F. Sabin proposed the most widely accepted theory about the origin of the lymphatic vasculature. This model proposed that isolated primitive lymph sacs bud from the endothelium of veins during early development; from these primary lymph sacs, the peripheral lymphatic system spreads by endothelial sprouting into tissues where local capillaries form. In 1999, we identified the homeobox gene Prox1 as the first specific marker of lymphatic endothelial cells. Functional inactivation of Prox1 in mice demonstrated that lymphangiogenesis requires the activity of this gene in a subpopulation of endothelial cells in embryonic veins. Prox1 promotes the development of the lymphatic vasculature by determining the final lymphatic fate of budding venous endothelial cells. On the basis of our findings, we propose a stepwise model of lymphangiogenesis in which lymphatic vasculature development is initiated by the specific expression of Prox1 in a subpopulation of vascular endothelial cells that subsequently adopt a lymphatic vasculature phenotype.