Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in regulating B cell receptor– and T cell receptor–mediated signaling. However, their role in natural killer (NK) cell development and functions is not well understood. Using mice expressing p110δ^D910A, a catalytically inactive p110δ, we show that these mice had reduced NK cellularity, defective Ly49C and Ly49I NK subset maturation, and decreased CD27^High NK numbers. p110δ inactivation marginally impaired NK-mediated cytotoxicity against tumor cells in vitro and in vivo. However, NKG2D, Ly49D, and NK1.1 receptor–mediated cytokine and chemokine generation by NK cells was severely affected in these mice. Further, p110δ^D910A/D910A NK cell–mediated antiviral responses through natural cytotoxicity receptor 1 were reduced. Analysis of signaling events demonstrates that p110δ^D910A/D910A NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation. These results reveal a previously unrecognized role of PI3K-p110δ in NK cell development and effector functions.