Immunosuppressive myeloid cells including myeloid-derived suppressor cells (MDSCs) contribute to multiple steps of cancer development. A better understanding of the molecular regulation of the functions of these myeloid cells and the signaling pathways will support development of novel anti-cancer therapeutic strategies. The leukocyte Ig-like receptor subfamily B (LILRB) proteins are a group of immune inhibitory receptors with intracellular immunoreceptor tyrosine-based inhibitory motifs. We have been studying the roles of these receptors in cancer development and immune regulation. The studies by us and others suggest that the LILRB family is becoming the next wave of myeloid immune checkpoint targets for cancer treatment. Here we demonstrated that LILRB3, a myeloid- specific member of this family, is functionally expressed on human MDSCs, and supports cancer development in mouse models. Importantly, we identified galectin-4 as an extracellular protein that binds to LILRB3 and induces LILRB3 activation, and the intracellular domain of LILRB3 interacts with the adaptor protein TRAF2 to contribute to NFκB upregulation. Furthermore, we developed anti-LILRB3 blocking antibodies that efficiently inhibit immunosuppressive activity of human MDSCs in vitro and cancer development in xenografted humanized mice and in LILRB3-transgenic mice. Our study suggests that LILRB3 represents an attractive novel target for cancer treatment. Based on new preliminary results, we propose the following Aims to test the hypothesis that LILRB3-initiated signaling in immunosuppressive myeloid cells supports cancer development. In Aim 1, we will determine the function of LILRB3 expressed on myeloid cells in cancer development. We will then determine whether galectin-4 regulates LILRB3- mediated signaling in tumor microenvironment to support cancer development in Aim 2. Finally we will dissect LILRB3 signaling in immunosuppressive myeloid cells in Aim 3. Our study will elucidate the molecular mechanisms by which LILRB3 regulates the activity of immunosuppressive myeloid cells, and lead to the development of innovative anti-cancer strategies based on targeting LILRB3 signaling.