Deep Springs Valley (DSV) is a hydrologically isolated valley between the White (north and west) and Inyo (south and east) Mountains that is commonly excluded from regional paleohydrologic and paleoclimate studies. Previous studies showed that uplift of Deep Springs ridge (informal name) by the Deep Springs fault defeated streams crossing DSV and hydrologically isolating the valley sometime after eruption of the Bishop Tuff. Here we present tephrochronology, clast counts, paleontology, and infrared stimulated luminescence (IRSL) data that reaffirms interruption of the Plio-Pleistocene hydrology and formation of DSV during the Pleistocene. Fossil gastropod, ostracodes, and charophytes along with IRSL dating document the 83.3-61.5 ka freshwater Deep Springs Lake, which roughly coincides with 71-57 ka Marine Isotope State 4 (MIS 4) glacial climate period. Documentation of the MIS-4 glacial climate in southwestern North America is sparse and pluvial Deep Springs Lake is indirect evidence of the MIS 4 glaciation that is corroborated by pluvial lakes in nearby Owens and Searles Valleys. We hypothesize that the MIS-4 Deep Springs Lake overflowed into Eureka Valley via the Soldier Pass wind gap. Hydrologic evolution of DSV has potential implications for understanding Pliocene and Pleistocene biotic dispersal pathways and endemism.