Natural Killer T (NKT) cells are a functionally diverse population that recognizes lipid‐based antigens in association with the antigen‐presenting molecule CD1d. Here, we define a technique to separate the functionally distinct thymic NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD278 (ICOS) and the activation‐associated glycoform of CD43, enabling the investigation of subset‐specific effector‐functions. We report that all three subsets express the transcription factor GATA‐3 and the potential to produce IL‐4 and IL‐10 following activation. This questions the notion that NKT2 cells are the predominant source of IL‐4 within the NKT cell pool, and suggests that IL‐10‐production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4⁺ and are biased toward Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll‐like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell‐biased response, even in the absence of exogenous antigen, and that combining LPS with α‐GalCer resulted in enhanced IL‐17A‐production, and reduced levels of the immunosuppressive cytokine IL‐10. This study provides a novel means to examine the context‐dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.