A TCRβ enhancer, known as the Eβ enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of Eβ during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the Eβ function. A single mutation at one of the two Runx binding motifs within the Eβ severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCRβ expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated Eβ activity, demonstrating that Runx complex binding is essential to initiate Eβ activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCRβ expression, whereas Eβ itself is continuously required for TCRβ expression. These findings imply that Runx complexes are essential for Eβ activation at the initiation phase, but are not necessary for maintaining Eβ activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for Eβ activity are differentially regulated, depending on the developmental stage and cellular activation status.