Objective To establish the cell models of c-myb+/+ and c-myb+/-ES in vitro by gene targeting with ES cell culture system, with the aim to examine the detailed role of the transcription factor c-myb in haemopoietic commitment and differentiation. Methods Haemopoietic differentiation was initiated by seeding ES cells in LIF-free methylcellulose medium. The embryoids of c-myb+/+ and c-myb+/-ES were analyzed by methylcellulose colony assay and real-time PCR, the formative process of embryoids and the expression of relative genes were compared in each haemopoietic system at different differentiation stages and procedures. Results The formative process of embryoids was similar for both c-myb+/+ and c-myb+/-ES cells, but the size and frequency of EBs were reduced in the case of the c-myb+/-cells. Similar kinetics gain existed for the formation of CFU-Es in both groups, but the number in c-myb+/-group was less than that of c-myb+/+ group, and the colonies were generally smaller. BFU-E was first detectable on day 7, and the peak value emerged on day 10 in both groups. Similar kinetics gain existed for the formation of CFU-M in the two groups, but the number was larger in c-myb+/-group than that in c-myb+/+ group, while the number of CFU-GM in c-myb+/-group was less than that in c-myb+/+ group. Real-time PCR analysis showed no changes on the gene expressions of ?-globin, ?-globin, GATA3, CD34, Lys, c-fms and c-mpl in both groups. Conclusions Sub-level expressions of c-myb (55%) and c-myb+/-are sufficient to allow progenitor to expand, but they throw a negative influence on the terminal differentiation. Sub-level expression of c-myb may influence the erythropoiesis and granulocytic development, but throw no influence on the precursor cells to differentiate into macrophages. The expressive levels of c-myb have no effect on the expressions of ?-globin, ?-globin, GATA3, CD34, Lys, c-fms and c-mp.