The host immune response to Mycobacterium tuberculosis (Mtb) plays a defining role in determining the clinical manifestations of tuberculosis disease and the ultimate outcome of infection. Phagocytic cells of the innate immune system, including macrophages, dendritic cells and neutrophils, are among the first cell types to encounter Mtb in the lung following aerosol transmission. The innate response provides a rapid and critical first line of defence against Mtb. Recent studies interrogating cellular diversity at a single cell level indicate that even within relatively homogeneous populations, sub-populations of innate immune cells exist with transcriptional profiles primed for differential responses to invading pathogens. To couple transcriptional differences to phenotypic differences in sub-populations of innate immune cells during Mtb infection, we are investigating the effects of Mtb and Type I, II and III interferons (IFNs) on gene expression in innate immune cell populations isolated from human peripheral blood using the PrimeFlow RNA assay (flow-FISH). Flow-FISH facilitates rare sub-population transcriptional profiling in parallel with conventional phenotypic flow cytometry, without the need for sorting specific cell subsets. By combining RNA detection with cell surface and intracellular protein expression, flow-FISH enables the identification of individual cells responding to a stimulus within a heterogeneous population. We have optimised the flow-FISH assay using in vitro-differentiated human monocyte-derived macrophages and dendritic cells, stimulated with Type I, II and III IFNs. We then compared these responses to whole blood stimulated ex vivo with IFNs, interrogating sub-populations of monocytes, dendritic cells and neutrophils, for differences in expression associated with phenotypic activation markers. Our data indicates heterogeneity in gene expression in response to IFN stimulation, even within in vitro-polarised cell populations. This work will provide new insights into the functional specialisation of innate immune cells in response to Mtb and distinct families of IFNs produced during Mtb infection.