Isolation and Functional Use of Human NK T Cells (Mark A. Exley andSteven P. Balk, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; S. Brian Wilson, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts). This unit describes methods for the isolation and functional analysis of human invariant NK T cells. The first protocol uses standard multicolor flow cytometry methods and staining reagents specific for V to generate invariant NK T cell clones. Support protocols for secondary stimulations and for rapid expansion of invariant NK T cell clones and functional analysis of these cells are also included.