P hosphatidylinositol-4,5-bisphosphate (PIP₂) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the α, β, and γ isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP₂ in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIγ^–/– megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIγ, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIβ- and PIP5KIγ^–/– cells had impaired PIP₂ synthesis. However, PIP5KIβ-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIβ in PIP5KIγ^–/– cells failed to revert this defect. Megakaryocytes lacking the PIP5KIγ-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIγ^–/– cells. These findings demonstrate a unique role for PIP5KIγ in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.