Despite their hematopoietic origin, mast cells (MCs) develop exclusively in tissues, hampering their ample use in research. To circumvent this problem, tissue-derived MCs are typically first expanded in culture, but the changes MCs may undergo in the novel micromilieu are poorly defined. Here, we monitor skin MCs from a number of donors over time, revealing profound yet non-synchronized modulations in culture. While tryptase and chymase, the most specific markers, strongly decline, FcεRI surface expression, and FcεRI-mediated histamine release steeply increase (from ≈15.5% to ≈60%), replicated by similar increments in TNF-α secretion. Interestingly, the modulations are independent of cell cycle progression, as they are comparable in the growth and postgrowth phase, implying they primarily result from microenvironmental conditioning. The data highlight a high degree of MC versatility, but also advise that results based on cultured MCs should be viewed with some caution, as they may not accurately reflect their counterparts in situ.