Abstract Disease outcome is known to be influenced by defined subsets of invariant NKT (iNKT) cells residing in distinct locations within peripheral tissue. However, the factors governing the development of these unique iNKT sublineages during thymic development are unknown. In this study we explored the mechanism by which E protein transcription factors and their negative regulators, the Id proteins, control the development of iNKT sublineages after positive selection. We found that E proteins directly bound the promyelocytic leukemia zinc finger (PLZF) promoter and were required for expression of this lineage-defining transcription factor and for the maturation and expansion of thymic iNKT cells. Moreover, expression of the negative regulators of E proteins, Id2 and Id3, defined distinct iNKT cell sublineages. Id3 was expressed in PLZFhigh NKT2 cells and loss of Id3 allowed for increased thymic iNKT cell expansion and abundance of the PLZF+ NKT2 sublineage. Id2 was expressed in T-BET+ NKT1 cells, and both Id proteins were required for the formation of this sublineage. Thus, we provide insight into E and Id protein regulation of iNKT cell proliferation and differentiation to specific sublineages during development in the thymus.