Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application towards dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune cells in mouse tumors and identified specific macrophage and conventional dendritic cell (cDC) subsets that are comparable to previously described human myeloid populations. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R selectively depleted macrophages with an inflammatory signature but spared a macrophage population that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated of a cDC1-Ccl22 population and gradually increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.