Although it is widely believed that invariant Va14⁺ NKT cells are generated from CD4⁺CD8⁺ double-positive (DP) thymocytes by selection on CD1d, a process termed the DP pathway, it is still not fully understood whether NKT cells develop exclusively by the DP pathway in a manner closely resembling that of conventional ab T cells, or some alternatives to this pathway exist. Here we provide genetic evidence supporting the presence of an alternative developmental pathway of NKT cells from late CD4^-CD8^- double-negative (DN) stage thymocytes, where the DP-specific ablation of Rag2 resulted in a loss of Va14Ja18 rearrangement by DP cells suggesting mature NKT cells developed in this mouse were generated from DN thymocytes with intact expressions of both Rag2 and Va14Ja18 transcripts. Moreover, fate-mapping approach, where a reporter gene expression is "turned on" starting from the DP stage, allowed detecting NKT cells that were differentiated through the DN pathway. Our present findings provide new insights in understanding the development of NKT cells in the thymus, which seems to be different from that of conventional ab T cells.