Updated publication reference for PubMed record(s): 31682227. Single-cell proteomics can reveal the changing protein composition of differentiating cells. We used shotgun mass spectrometry to determine the abundant proteins present in single or small pools of subpicoliter-sized cells from the embryonic day 15 (E15) utricle of the chicken inner ear, when many hair cells are differentiating from progenitor (supporting) cells. The actin monomer binding protein thymosin β4 (TMSB4X) was present in E15 progenitor cells at nearly equimolar levels relative to actin, but dropped to one-tenth that value in hair cells, with little change in total actin. Single-cell RNA-seq analysis of E15 utricle cells showed that TMSB4X transcripts fell in abundance once hair-cell differentiation initiated. These results suggest that most actin is sequestered in progenitor cells, but upon differentiation to hair cells, actin is released, permitting assembly of the sensory hair bundle.