Thrombospondin-1 (TSP-1) is a potent anti-angiogenic factor downregulated in many tumors. CD36 and β1-integrin are two of its key receptors, mediating its anti-angiogenic activity by initiating signaling cascades that inhibit endothelial cell migration and promote apoptosis. Receptor clustering on the plasma membrane is thought to be important for initiating these signaling cascades. However, little is known about the mechanisms that contribute to CD36 and β1-integrin clustering and how they lead to downstream signals. In this study, we used quantitative single-molecule and super-resolution imaging to measure the dynamics and spatial organization of CD36 and β1-integrin in human microvascular endothelial cells (HMVECs). We compared receptor dynamics and spatial organization between unstimulated cells and cells exposed to TSP-1 or 3TSR, a small subdomain of TSP1 which primarily binds to CD36 and β1-integrin. We found that treatments with either TSP-1 or 3TSR, at doses that lead to HMVEC apoptosis, result in a significant increase in CD36 mobility in a β1-integrin-dependent manner. We also found that treatment with TSP-1 increases Src phosphorylation at focal adhesions in a CD36 dependent manner. Based on our data, we propose that CD36 and β1-integrin work together to initiate cellular signaling downstream of TSP-1 binding.