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In response to pheromone, many proteins localize on the plasma membrane of yeast cell to reform it into a polarized shmoo structure. The adaptor protein Ste50p, known as a pheromone signal enhancer critical for shmoo polarization, has never been explored systematically for its localization and function in the polarization process. Time-lapse single-cell imaging and quantitation shown here characterizes Ste50p involvement in the establishment of cell polarity. We found that Ste50p patches on the cell cortex mark the point of shmoo initiation, these patches could move, and remain associated with the growing shmoo tip in a pheromone concentration time-dependent manner until shmoo maturation. A Ste50p mutant impaired in patch localization suffers a delay in polarization. By quantitative analysis we show that polarization correlates with the rising levels of Ste50p, enabling rapid cell responses to pheromone that correspond to a critical level of Ste50p at the initial G1 phase. We exploited the quantitative differences in the pattern of Ste50p expression to correlate with the cell-cell phenotypic heterogeneity, showing Ste50p involvement in the cellular differentiation choice. Taken together, these findings present Ste50p to be part of the early shmoo development phase, suggesting that Ste50p may be involved with the polarisome in the initiation of polarization, and plays a role in regulating the polarized growth of shmoo during pheromone response.