Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP₃) and phosphatidylinositol-3,4-bisphophate (PI34P₂), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP₃ and PI34P₂ in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P₂ selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP₃ selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP₃ and PI34P₂ and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.