Summary. Background: We have previously shown that the receptor-like protein tyrosine phosphatase (PTP) CD148 is essential for initiating glycoprotein VI (GPVI) signaling in platelets. We proposed that CD148 does so by dephosphorylating the C-terminal inhibitory tyrosine of Src family kinases (SFKs). However, this mechanism is complicated by CD148-deficient mouse platelets having a concomitant reduction in GPVI expression. Objectives: To investigate the effect of CD148 on GPVI signaling independent of the decrease in GPVI expression and to further establish the molecular basis of the activatory effect of CD148 and downregulation of GPVI. Methods: CD148-deficient mouse platelets were investigated for functional and biochemical defects. The DT40/NFAT-lucifierase reporter assay was used to analyze the effect of CD148 on GPVI signaling. CD148–SFK interactions and dephosphorylation were quantified using biochemical assays. Results: CD148-deficient mouse platelets exhibited reduced collagen-mediated aggregation, secretion and spreading in association with reduced expression of GPVI and FcR γ-chain and reduced tyrosine phosphorylation. The phosphorylation status of SFKs suggested a global reduction in SFK activity in resting CD148-deficient platelets. Studies in a cell model confirmed that CD148 inhibits GPVI signaling independent of a change in receptor expression and through a mechanism dependent on tyrosine dephosphorylation. Recombinant CD148 dephosphorylated the inhibitory tyrosines of Fyn, Lyn and Src in vitro, although paradoxically it also dephosphorylated the activation loop of SFKs. Conclusions: CD148 plays a critical role in regulating GPVI/FcR γ-chain expression and maintains a pool of active SFKs in platelets by directly dephosphorylating the C-terminal inhibitory tyrosines of SFKs that is essential for platelet activation.