Abstract Spectral flow cytometry is an advanced immunological tool that can enable comprehensive analysis of the immune system by simultaneously comparing innate and adaptive immune cells. Here, using a 40‐color antibody panel, we advance our knowledge of innate‐like cells by investigating chemokine receptors and maturation markers not usually assessed on these populations, examining age‐related effects on these immune cell subsets. We characterize phenotypic changes of peripheral blood mononuclear cells (PBMCs) in three age groups: newborns (cord blood), adults aged 20–30 years, and adults aged > 70 years. We compare the age‐related changes of innate cells, including ILCs, NK cells, monocytes, dendritic cells, and innate‐like T cells, comparing them with memory T cells. We also examine subsets of CD4 − CD8 − double‐negative (DN) T cells and CD3 + CD161 + T cells, revealing they are phenotypically similar to known subsets of innate‐like T cells, and they also increase in frequency in an age‐related manner. The frequencies of ILC1 increased with age, ILC2 remained stable, whereas ILC3 peaked in young adults, and were higher than cord and older adults. Notably, we identify the NK cell maturation marker, CD57, as a universal marker that defines aging populations of both innate and adaptive immune cells. This study enhances our understanding of the ontogeny of human immune cells, highlighting significant age‐related changes in the frequency and phenotype of innate‐like populations of immune cells.