RUNX1 is essential for definitive hematopoiesis and T‐cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1‐2A, carrying two phospho‐deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1‐4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T‐cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1‐deficient cells. Notably, RUNX1‐5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho‐mimic proteins RUNX1‐4D/E and RUNX1‐5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis.