The lymphatic vasculature originates from the blood vasculature through a mechanism relying on Prox1 expression and VEGFC signalling, and is separated and kept separate from the blood vasculature in a Syk- and SLP76-dependent manner. However, the mechanism by which lymphatic vessels are separated from blood vessels is not known. To gain an understanding of the vascular partitioning, we searched for the affected gene in a spontaneous mouse mutant exhibiting blood-filled lymphatic vessels, and identified a null mutation of the Plcg2 gene, which encodes phospholipase Cγ2 (PLCγ2),by positional candidate cloning. The blood-lymph shunt observed in PLCγ2-null mice was due to aberrant separation of blood and lymphatic vessels. A similar phenotype was observed in lethally irradiated wild-type mice reconstituted with PLCγ2-null bone marrow cells. These findings indicate that PLCγ2 plays an essential role in initiating and maintaining the separation of the blood and lymphatic vasculature.