Anergic T cells have altered diacylglycerol metabolism, but whether that altered metabolism has a causative function in the induction of T cell anergy is not apparent. To test the importance of diacylglycerol metabolism in T cell anergy, we manipulated diacylglycerol kinases (DGKs), which are enzymes that terminate diacylglycerol-dependent signaling. Overexpression of DGK-α resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-α-deficient mice and found that DGK-α-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-α-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-α or DGK-ζ proliferated and produced interleukin 2. Pharmacological inhibition of DGK-α activity in DGK-ζ-deficient T cells that received an anergizing stimulus proliferated similarly to wild-type T cells that received CD28 costimulation and prevented anergy induction. Our findings suggest that regulation of diacylglycerol metabolism is critical in determining whether activation or anergy ensues after T cell receptor stimulation.