This study describes a new approach to discern early divergence in stem cell lineage progression via temporal dynamics of the cytoskeletal protein, F-actin. The approach involves real-time labeling of human mesenchymal stem cells (MSCs) and longitudinal tracking of the turnover dynamics of a fluorogenic F-actin specific probe, SiR-actin (SA). Cells cultured in media with distinct lineage factors and labeled with SA showed lineage specific reduction in the actin turnover shortly after adipogenic (few minutes) and chondrogenic (3-4 hours) commitment in contrast to osteogenic and basal cultured conditions. Next, composite staining of SA along with the competing F-actin specific fluorescent conjugate, phalloidin, and high-content image analysis of the complementary labels showed clear phenotypic parsing of the sub-populations as early as 1-hour post-induction across all three lineages. Lastly, the potential of SA-based actin turnover analysis to distinguish cellular aging was explored. In-vitro aged cells were found to have reduced actin turnover within 1-hour of simultaneous analysis in comparison to cells of earlier passage. In summary, SiR-actin fluorescent reporter imaging offers a new platform to sensitively monitor emergent lineage phenotypes during differentiation and aging and resolve some of the earliest evident differences in actin turnover dynamics.