The transcription factor RUNX3 is a prominent regulator of multiple hematopoietic cell lineages. Gene loss of function studies demonstrated the unique and essential roles of this master regulator in differentiated lymphoid and myeloid cells. As a complementary approach, RUNX3 was upregulated in various leukocyte subsets to probe the instructive role of this 'multilineage'-specific transcription factor. In this report, we overview the immunomodulatory functions of RUNX3 within the hematopoietic compartment to gain insight into the consequences of Runx3 deletion or overexpression in committed immune cells. Genetic studies revealed the essential role of RUNX3 in Langerhans cell development. Moreover, this transcription factor is necessary for the differentiation and maintenance of the cytotoxic CD8+ T cells. In addition, T helper, natural killer, and B cells are also influenced by RUNX3. Importantly, the ectopic expression of Runx3 enhances the immunogenicity of cytotoxic T cells and pluripotent stem-cell-derived dendritic cells, suggesting that this protein can be applied in cell-based immunotherapies.