We previously identified apoptosis stimulating protein of p53 (Aspp1) as an endothelial-specific gene functioning in mouse embryogenesis. To investigate the in vivo role of Aspp1, we generated Aspp1 knockout mice by targeted disruption. Aspp1^−/− embryos showed subcutaneous edema and disorganized lymphatic vasculature. Morphological changes in lymphatic endothelial cells and isolated lymphatic islands were detected in Aspp1^−/− embryos. Lymphangiography by injecting dye subcutaneously into the embryonic forelimb showed defective lymphatic drainage function and obstruction in collecting lymphatic vessels of Aspp1^−/− embryos. Interestingly, Aspp1^−/− adult mice resolved these lymphatic functional defects seen during embryogenesis, but lymphangiography in Aspp1^−/− adult mice revealed abnormal patterns in collecting lymphatic vessels. Since Aspp proteins reportedly enhance apoptotic activity of p53, we asked whether p53 deficiency also affected lymphatic vessel development. Analysis of p53 knockout or Aspp1; p53 double knockout mice showed that p53 loss did not affect lymphatic vessels. These results indicate that Aspp1 plays a crucial role in the initial assembly and function of lymphatic vessels during mouse development in a p53-independent manner. Here we report novel lymphatic vascular phenotypes in Aspp1^−/− mice; subcutaneous edema detected only during embryogenesis, delayed lymphatic vessel formation, and mispatterned collecting lymphatic vessels.