Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help.This chapter describes the basic procedures that can be used to design a T cell cloning experiment. A prototypic limiting dilution cloning procedure to obtain and maintain in culture peptide-specific CD4 positive T cell clones from peripheral blood is detailed. Methods to generate other types of T cell clones are described in Section 3.5. Since peripheral blood is not always the ideal source of lymphocytes to isolate T cell clones, in Section 3.5 the isolation of cells from biopsies or other tissues is briefly described.Finally, in the notes, some expedients and wariness that can be useful to improve the efficiency of T cell cloning are reported.