The critical role of costimulatory signals in T-cell activation and the complexity of T-cell costimulatory pathways involved make a detailed understanding of this system a challenging task. By taking advantage of the unique chemical properties of CFSE, we and others have developed an in vivo model that allows quantitative analysis of T-cell activation at a single-cell level. This model involves labeling of donor T-cells with the tracking dye CFSE and adoptively transferring into lethally irradiated allogeneic hosts. T-cells proliferating in the host mice can be explicitly analyzed upon recovery. By using mice deficient for certain costimulatory molecules as a source of donor cells or by treating the host mice with reagents that block certain costimulatory pathways, this CFSE model is extremely useful in studying the role of T-cell costimulatory signals in activation, survival, and effector differentiation of alloreactive T-cells in vivo.