Although protein S-palmitoylation was first characterized >30 years ago, and is implicated in the function, trafficking, and localization of many proteins, little is known about the regulation and physiological implications of this posttranslational modification. Palmitoylation of various signaling proteins required for TCR-induced T cell activation is also necessary for their proper function. Linker for activation of T cells (LAT) is an essential scaffolding protein involved in T cell development and activation, and we found that its palmitoylation is selectively impaired in anergic T cells. The recent discovery of the DHHC family of palmitoyl acyl transferases and the establishment of sensitive and quantitative proteomics-based methods for global analysis of the palmitoyl proteome led to significant progress in studying the biology and underlying mechanisms of cellular protein palmitoylation. We are using these approaches to explore the palmitoyl proteome in T lymphocytes and, specifically, the mechanistic basis for the impaired palmitoylation of LAT in anergic T cells. This chapter reviews the history of protein palmitoylation and its role in T cell activation, the DHHC family and new methodologies for global analysis of the palmitoyl proteome, and summarizes our recent work in this area. The new methodologies will accelerate the pace of research and provide a greatly improved mechanistic and molecular understanding of the complex process of protein palmitoylation and its regulation, and the substrate specificity of the novel DHHC family. Reversible protein palmitoylation will likely prove to be an important posttranslational mechanism that regulates cellular responses, similar to protein phosphorylation and ubiquitination.