Perivascular supporting cells, including vascular smooth muscle cells (VSMC) and pericytes (PC), provide instructive signals to adjacent endothelial cells (EC) helping to maintain vascular homeostasis. These signals are provided through direct contact and by release of soluble factors by these cells. Thrombospondin-1 (TSP1) is a matricellular protein and an autocrine factor for VSMC. TSP1 activity along with that of platelet-derived growth factor (PDGF) regulates VSMC proliferation and migration. However, the manner in which TSP1 and PDGF impact retinal PC function requires further investigation. Here we describe, for the first time, the isolation and culture of retinal PC from wild type (TSP1+/+) and TSP1-deficient (TSP1-/-) immortomice. We show these cells express early and mature markers of PC including NG2, PDGF-receptor (PDGF-R), and smooth muscle actin, as well as desmin, calbindin, and mesenchymal stem cell markers. These cells were successfully passaged and maintained in culture for several months without significant loss of expression of these markers. TSP1+/+ PC proliferated at a faster rate compared to TSP1-/- PC. In addition, TSP1+/+ PC, like VSMC, responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1-/- PC failed to respond to the promigratory and proliferative activity of PDGF-BB. This may be attributed, at least in part, to the limited interaction of PDGF-BB with TSP1 in null cells, which is essential for PDGF proliferative and migratory action. We observed no significant differences in the rates of apoptosis in these cells. TSP1-/- PC were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and v3 integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis.