Phosphatidylinositol-4, 5-bisphosphate (PIP2) is vital for the signaling cascades that are critical for actin dynamics. Although all PIP5K isoforms (α , β , and γ ) synthesize PIP2 by phosphorylating PI4P, the isoforms have different primary structures, expression levels in various tissues, and intracellular localization. To test the hypothesis that the functions of these isoforms are also different, we have generated murine lines that contain null mutations in either of two platelet PIP5K isoforms, PIP5Kβ and PIP5Kγ . PIP5Kβ -null mice were born 42% less than anticipated by Mendelian genetics, and heterozygotes were born 18% less than the expected frequency. Adult PIP5Kβ −/− mice had normal platelet counts and exhibited no spontaneous hemorrhage. However, PIP5Kβ −/− platelets had impaired aggregation in response to thrombin and thromboxane analogues. In addition, following agonist stimulation they failed to properly synthesize additional PIP2. Platelets lacking PIP5Kβ also exhibited a marked defect in spreading upon immobilized fibrinogen. This failure to spread was associated with a striking decrease in filopodia formation and extension of actin rich lamellipodia. In contrast to the phenotype of PIP5Kβ -knockout mice, we found that targeted disruption of PIP5Kγ results in early prenatal mortality due to a cardiovascular developmental defect. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. Similar to PIP5Kβ knockout megakaryocytes, PIP5Kγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we examined membrane dynamics during cell adhesion in real time. Wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ -null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. Using laser tweezers to pull the cell membrane apart from the cytoskeleton, we found that long tethers of the membrane could easily be drawn from PIP5Kγ −/− megakaryocytes, but not from PIP5Kγ +/− or PIP5Kβ −/− megakaryocytes. This implies that PIP5Kγ −/− megakaryocytes have a defect in their ability to anchor the cell membrane to the cytoskeleton. This defect was attributable to the ability of PIP5Kγ to synthesize PIP2 since it could be rescued by adding back wild type PIP5Kγ , but could not be rescued by a catalytically inactive PIP5Kγ mutant. Accordingly, our data are consistent with the hypothesis that different PIP5K isoforms contribute to compartmentalized pools of PIP2 that contribute to different aspect of platelet &megakaryocyte actin dynamics. PIP5Kβ synthesizes PIP2 that contributes to the formation of filopodia and lamellipodia, while PIP5Kγ generates PIP2 that is required for the stable association of the membrane with the cytoskeleton.