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Abstract Marine Isotopic Stage 5 is associated with wetter climatic conditions in the Saharo-Arabian deserts. This stage also corresponds to the establishment of Middle Paleolithic hominins and their associated material culture in two geographical provinces in southwest Asia—the Eastern Mediterranean woodland and the Arabian Peninsula desert. The lithic industry of the Eastern Mediterranean is characterized by the centripetal Levallois method, whereas the Nubian Levallois method characterizes the populations of the Arabian desert. The Negev Desert, situated between these regions is a key area to comprehend population movement in correlation to climatic zones. This investigation addresses the nature of the Middle Paleolithic settlement in the Negev Desert during MIS 5 by studying the site of Nahal Aqev. High resolution chronological results based on luminescence dating and cryptotephra show the site was occupied from MIS 5e to MIS 5d. The lithic industries at Nahal Aqev are dominated by centripetal Levallois core method. These data demonstrate that Nahal Aqev is much closer in its cultural attributes to the Eastern Mediterranean Middle Paleolithic than to the Arabian Desert entity. We conclude that Nahal Aqev represents an expansion of Middle Paleolithic groups from the Mediterranean woodland into the desert, triggered by better climatic conditions. These groups possibly interacted with hominin groups bearing the Nubian core tradition from the vast region of Arabia.