Current research on antigen specific CD4+ T cells indicates that there is functional and phenotypic heterogeneity within these populations, but the extent of this heterogeneity is poorly described. The CD134/CD25 assay allows live isolation of antigen specific cells in vitro for down-stream molecular analysis. Antigen specific CD4+ T cells were examined at the molecular level by lineage specific transcription factor profiling using qualitative multiplex single cell RT-PCR and Lock Nucleic Acid (LNA) probes allowed unbiased amplification and delineation of expression of Tbx21, Gata3, Rorc, Foxp3 and Bcl-6. It overcomes the limitations of previous assays by allowing identification of transcription factor mRNA in single antigen specific cells with high sensitivity (down to 10 femtograms) and specificity. Patterns of responses can be robustly characterized using <200 cells based on exact binomial calculations. These results are reproducible with a CV of ≈6%. The patterns of heterogeneity are stable within an individual antigen specific response but vary between responses to different antigens. Responses to CMV have a Th1 predominant profile (35.6% of responding cells expressing tbx21) whereas responses to Tetanus Toxoid have a Th2 biased profile (22% of responding cells expressing gata3), with unexpectedly high levels of Treg cells found in both populations. Here we describe a methodology that allows live isolation of Ag specific cells and transcription factor profiling at a single cell level to robustly delineate the different CD4+ T cell subsets within this population. This novel method is a powerful tool that can be used to study CD4+ T cell heterogeneity within extremely small populations of cells and where cell numbers are limited.