The origin of the Middle Stone Age (MSA) marks the transition from a highly persistent mode of stone toolmaking, the Acheulean, to a period of increasing technological innovation and cultural indicators associated with the evolution of Homo sapiens. We used argon-40/argon-39 and uranium-series dating to calibrate the chronology of Acheulean and early MSA artifact-rich sedimentary deposits in the Olorgesailie basin, southern Kenya rift. We determined the age of late Acheulean tool assemblages from 615,000 to 499,000 years ago, after which a large technological and faunal transition occurred, with a definitive MSA lacking Acheulean elements beginning most likely by similar to 320,000 years ago, but at least by 305,000 years ago. These results establish the oldest repository of MSA artifacts in eastern Africa.