Abstract Innate immune cells, including macrophages and dendritic cells, protect the host from pathogenic assaults in part through secretion of a program of cytokines and chemokines (C/Cs). Cell-to-cell variability in C/C secretion appears to contribute to the regulation of the immune response, but the sources of secretion variability are largely unknown. To begin to track the biological sources that control secretion variability, we developed and validated a microfluidic device to integrate live-cell imaging of fluorescent reporter proteins with a single-cell assay of protein secretion. We used this device to image NF-κB RelA nuclear translocation dynamics and Tnf transcription dynamics in macrophages in response to stimulation with the bacterial component lipopolysaccharide (LPS), followed by quantification of secretion of TNF, CCL2, CCL3, and CCL5. We found that the timing of the initial peak of RelA signaling in part determined the relative level of TNF and CCL3 secretion, but not CCL2 and CCL5 secretion. Our results support evidence that differences in timing across cell processes partly account for cell-to-cell variability in downstream responses, but that other factors introduce variability at each biological step.