HIV-1 assembles at the plasma membrane (PM) of infected cells. PM association of the main structural protein Gag depends on its myristoylated MA domain and PM PI(4,5)P2. Using a novel chemical biology tool that allows rapidly tunable manipulation of PI(4,5)P2 levels in living cells, we show that depletion of PI(4,5)P2 completely prevents Gag PM targeting and assembly site formation. Unexpectedly, PI(4,5)P2 depletion also caused loss of pre-assembled Gag lattices from the PM. Subsequent restoration of PM PI(4,5)P2 reinduced assembly site formation even in the absence of new protein synthesis, indicating that the dissociated Gag molecules remained assembly competent. These results reveal an important role of PI(4,5)P2 for HIV-1 morphogenesis beyond Gag recruitment to the PM and suggest a dynamic equilibrium of Gag-lipid interactions. Furthermore, they establish an experimental system that permits synchronized induction of HIV-1 assembly leading to induced production of infectious virions by targeted modulation of Gag PM targeting.