The induction of pancreatic endocrine differentiation from undifferentiated precursor cells is a multi-step process regulated by the expression of several transcription factors. At E9.5 expression of homeodomain protein Pdx-1 defines the early pancreatic epithelium. Later, expression of the basic helix-loop-helix factor, Neurogenin 3 (Ngn3) marks the initiation of the endocrine lineage from the pancreatic precursor cells. A full understanding of these processes is essential in order to control development of insulin secreting beta-cells from embryonic stem cells in vitro. Since the expression of ngn3 is a key step, the aim of this work was to develop monoclonal antibodies for immunohistochemical (IHC) detection of Ngn3. All mice (RBF-strain) immunized with recombinant GST-mNGN3 fusion protein responded with a high antibody titer. The sera were further screened by IHC on paraffin sections of fetal (E13) mouse pancreas, and two hybridomas subsequently derived from one selected mouse produced antibodies with prominent and specific Ngn3 staining properties. These new antibodies provide novel useful tools for co-labeling studies using the large panel of existing rabbit polyclonal antibodies available against important transcription factors to further characterize the development of pancreatic endocrine progenitor cells.