Complex interdisciplinary studies carried out in the territory of the Vilnius Lower Castle, E Lithuania, were used to construct a chronological framework based on radiocarbon data and archaeological information. Bulk samples (wood and sediment) were collected from an approximately 3-m core that crossed cultural layers and underlying strata. 14C dates indicate that the underlying bed possibly formed during the 6th century AD, although no archaeological finds were discovered there. Paleobotanical (pollen and plant macrofossil) investigations reveal evidence of agriculture that points to the existence of a permanent settlement in the area at that time. The chronological data indicates a sedimentation hiatus before the onset of the deposition of the cultural layer in the studied area. The 14C dates showed that the formation of the cultural bed began during the late 13th–early 14th centuries AD, that is, earlier than expected according to the archaeological record. The ongoing deposition of the cultural beds continued throughout the middle to latter half of the 14th century AD as revealed by the archaeological records and confirmed by well-correlated 14C results. After some decline in human activity in the middle of the 14th century AD, a subsequent ongoing development of the open landscape, along with intensive agriculture, points to an increase in human activity during the second half of the 14th century AD. The first half of the 15th century AD was marked by intensive exploitation of the territory, indicating a period of economic and cultural prosperity. The chronological framework indicates that the investigated cultural beds continued forming until the first half of the 16th century AD.