Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signalling proteins in regulating MK migration and platelet recovery following immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow-derived MKs on a fibronectin matrix are abolished by the Src family kinases (SFKs) inhibitor PP1, the Syk inhibitor R406 and the integrin alphaIIbbeta3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary PLCgamma2-deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the GPVI-FcRgamma-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of PECAM-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCgamma2. This study identifies a model in which integrin signalling via SFKs and Syk kinase to PLCgamma2 is required for MK spreading, migration and platelet formation.