Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction in the order of 65-75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes by conventional and immunoelectron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were under-represented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in megakaryocytes underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.