Whilst pre-mRNA splicing has been demonstrated to play functional roles in normal hematopoiesis, the potential importance of many splicing regulators remains unexplored. RNA-binding motif protein 25, (RBM25), is a splicing factor involved in multiple cellular functions, such as proliferation and apoptosis, in various tissues as well as in leukemia. Here, we use a conditional knock-out model to show that the fundamental role of RBM25 in alternative splicing is reflected in the pivotal role of the protein for multiple hematopoietic lineages, including long-term hematopoietic stem cells, as well as embryonic stem cells derived from gene targeted mice. In contrast, mono-allelic deletion of Rbm25 did not impair HSC self-renewal or differentiation, neither under steady-state conditions nor after proliferative stress induced by bone marrow transplantation. Thus, we demonstrate that Rbm25 is haplosufficient and required for the maintenance of normal murine hematopoiesis.