To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+ CD27+, the CCR7− CD27+, and the CCR7− CD27− population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+ CD27+ to CCR7− CD27+ to CCR7− CD27−. Telomere length in these subsets differed significantly (CCR7+ CD27+> CCR7− CD27+> CCR7− CD27−), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+ CD27+, whereas IFN-γ was produced by CCR7− CD27+ and to a slightly lesser extent by CCR7− CD27− T cells. IL-4 secretion was predominantly conducted by CCR7− CD27− memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.