The mitogen-activated protein kinases (MAPKs) ERK1/2, p38, and JNK are thought to determine survival-versus-death fate in developing thymocytes. However, this view was challenged by studies using 'MEK1-ERK1/2-specific' pharmacological inhibitors, which block both positive and negative selection. Recently, these inhibitors were also shown to affect MEK5, an upstream activator of ERK5, another class of MAPK with homology to ERK1/2. To define the contribution of the MEK5-ERK5 pathway in T-cell development, we retrovirally expressed dominant-negative or constitutively activated form of MEK5 to inhibit or activate the MEK5-ERK5 pathway. We demonstrate that MEK5 regulates apoptosis of developing thymocytes but has no function in positive selection. ERK5 activity correlates with the levels of Nur77 family members but not that of Bim, two effector pathways of thymocyte apoptosis. These results illustrate the critical involvement of the MEK5-ERK5 pathway in thymocyte development distinct from that of ERK1/2 and highlight the importance of the MAPK network in mediating differential effects pertaining to T-cell differentiation and apoptosis.