In longitudinal clinical studies, receiving a high percentage of allogeneic donor-derived CD4+CCR7+ T cells, which include naïve and central memory subsets have been correlated with increased incidence and severity of acute GVHD. Whether naïve and central memory CD4+ T-cell subsets contribute more or equally to alloimmune responses are still unclear in human. The aim of this study was to investigate in vitro the alloreactive response of purified naïve, central memory, and effector memory CD4+ T-cell subsets in HLA identical setting. By coculturing monocyte-derived dendritic cells and purified CD4+ T-cell subsets, from healthy HLA-identical male and female sibling pairs, we found that naïve CD4+CCR7+CD45RA+ T cells developed the highest proliferative response upon stimulation by minor histocompatibility antigens and were progressively driven to produce high levels of interferon-γ, tumor necrosis factor, and interleukin-6. Comparatively, the central memory CD4+CCR7+CD45RAneg subset proliferated to a lower extent and produced very low amounts of pro-inflammatory cytokines while the CCR7neg effector memory CD4+ subset was unresponsive. This study demonstrates the superior capacity of naïve CD4+ T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro-inflammatory differentiation makes them potentially acute GVHD inducers. These in vitro results in line with what we have observed in clinical studies and may also lend support to approaches of partial selective T-cell depletion for GVHD prevention.