AbstractThe mammalian Pre‐B cell leukaemia transcription factors 1–4 (PBX1‐4) constitutes the PBC class of the homeodomain (HD)‐containing proteins, which play important roles in diverse developmental processes. The functions and the underlying molecular mechanisms of PBX1‐3 but not PBX4 have been extensively studied, and they have been reported to direct essential morphogenetic processes and organogenesis. In the present study, we generated knockin mice of FLAG‐tagged PBX4 and the Pbx4 knockout (KO) mice and carried out in‐depth characterisation of PBX4 expression and function. PBX4 was initially detected only in the testis among several organs of the adult mice and was expressed in spermatocytes and spermatids. However, no abnormality in spermatogenesis, but growth retardation and premature death after birth were observed in most adult Pbx4 KO mice. These animals were inactive and had shorter hindlimbs and lower numbers of reticulocytes and lymphocytes, probably caused by abnormalities at earlier developmental stages. Pbx4 mRNAs were indeed detected in several embryonic cell types related to limb development by in situ hybridisation and single‐cell RNA‐sequencing analysis. Pbx4 protein was also detected in the bone marrow of adult mice with a lower level compared with that in the testis. PBX4 preferentially binds to the promoters of a large number of genes including those for other HD‐containing proteins and ribosomal proteins whose mutations are related to anaemia. PBX4‐binding sites are enriched in motifs similar to those of other HD‐containing proteins such as PKNOX1 indicating that PBX4 may also act as a co‐transcription factor like other PBC proteins. Together, these results show that PBX4 participates in limb development and haematopoiesis while its function in spermatogenesis has not been revealed by gene KO probably due to the complementary effects of other genes.