In this study, we describe the use of intravital microscopy in a transgenic mouse model expressing yellow fluorescent protein (YFP) under the control of a monocyte specific promoter c-fms (CD115) to track and quantify specific leukocyte subsets. Flow cytometry on peripheral and bone marrow leukocytes revealed that YFP was predominantly expressed by CD11a + , CD11b + , and CD14 + monocytes. In the bone marrow, 67±4% of Ly6C high F4/80 + cells were YFP high while 55±1% of Ly6C low F4/80 + cells were YFP low supporting the use of c-fms YFP expression as a marker of monocyte lineage. 70±7% of CD11b + F4/80 + Ly6C + (“triple positive”) cells expressed YFP. To assess leukocyte–endothelial interactions in YFP + cells in c-fms YFP+ mice, we evaluated leukocyte adhesion, rolling and local shear stress responses in the cremasteric endothelium 4 h following administration of TNFα. TNFα resulted in a five-fold increase in adhesion of YFP + cells to the endothelium and provided superior discriminative ability in assessing rolling and adhesion events when compared with bright field microscopy. Additionally, when compared with Rhodamine-6G labeled leukocytes or GFP + cells in mice transplanted with green fluorescent protein (GFP) positive bone marrow, the level of detail observed in the c-fms YFP+ was greater, with both GFP + and YFP + cells demonstrating superior signal to noise compared to bright field microscopy. A weak positive linear correlation between wall shear stress and YFP + cell adhesion (r 2 =0.20, p<0.05) was seen in the cremasteric microcirculation. Taken together, these data demonstrate the use of c-fms YFP+ mice in identifying distinct monocyte subsets and highlight the potential of this model for real-time monocyte–endothelial interactions using intravital microscopy.