The three isoforms of PIP5KI (α, β, and γ) synthesize PI4,5P2 (PIP2) by phosphorylating PI4P. Therefore, it is not clear why platelets, like all eukaryotic cells, have more than one isoform. To test the hypothesis that PIP5KI isoforms have nonoverlapping functions, we generated a murine line containing a null mutation of PIP5KIβ and analyzed the effect on platelet signaling. PIP5KIβ-null mice had normal platelet counts. In contrast to platelets lacking PIP5KIα, platelets lacking PIP5KIβ exhibited impaired aggregation accompanied by disaggregation. Although platelets lacking PIP5KIβ had only a moderate deficiency of PIP2 under basal conditions, they had a striking deficiency in PIP2 synthesis and IP3 formation after thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP3 synthesis even though they still contain PIP5KIγ, the predominant PIP5KI isoform in platelets. These results demonstrate that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP2 that is required for second-messenger formation, whereas the pool of PIP2 synthesized by PIP5KIγ does not contribute to this process. Additionally, we found that PIP5KIβ-null platelets failed to form arterial thrombi properly in vivo. Together, these data demonstrate that PIP5KIβ is required for rapid PIP2 synthesis, second-messenger production, and stable platelet adhesion under shear in vivo. These results also demonstrate that after stimulation of a G protein-coupled receptor, IP3 is completely derived from a rapidly synthesized discrete pool of PIP2 synthesized by PIP5KIα and PIP5KIβ. © 2008 by The National Academy of Sciences of the USA.