Mature megakaryocytes form structures called proplatelets that serve as conduits for platelet packaging and release at vascular sinusoids. Since the megakaryocyte expresses abundant levels of integrin α_IIbβ₃, we have examined a role for fibrinogen in proplatelet development and platelet release alongside that of other matrices. Primary mature murine megakaryocytes from bone marrow aspirates readily formed proplatelets when plated on fibrinogen at a degree that was significantly higher than that seen on other matrices. In addition, α_IIbβ₃ was essential for proplatelet formation on fibrinogen, as megakaryocytes failed to develop proplatelets in the presence of α_IIbβ₃ antagonists. Interestingly, inhibition of Src kinases or Ca²⁺ release did not inhibit proplatelet formation, indicating that α_IIbβ₃-mediated outside-in signals are not required for this response. Immunohistochemical studies demonstrated that fibrinogen is localized to the bone marrow sinusoids, a location that would allow it to readily influence platelet release. Further, thrombopoietin-stimulated α_IIb^-/- mice had a reduced increase in platelet number relative to controls. A similar observation was not observed for platelet recovery in α_IIb^-/- mice in response to antibody-induced thrombocytopenia, indicating the existence of additional pathways of regulation of proplatelet formation. These results demonstrate that fibrinogen is able to regulate proplatelet formation via integrin α_IIbβ₃.