Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.