Dendritic cells (DC) are professional antigen-presenting cells capable of initiating primary immune responses. They have been intensively studied and are used in both basic immunology research and clinical immunotherapy. However, the genetic pathways leading to DC differentiation and maturation remain poorly understood. Using focused microarrays with oligonucletotide probes for 120 genes encoding co-stimulatory molecules, chemokines, chemokine receptors, cytokines, cytokine receptors, TLRs, and several other related molecules, we analyzed the kinetics of gene expression for the overall differentiation process of monocytes into mature DC. In parallel, we compared the transcriptional profiles in DC maturation in the presence of LPS, TNF-alpha or trimeric CD40L. We found similar transcriptional profiles for early immature DC and immature DC, respectively generated by culturing monocytes with GM-CSF and IL-4 for three or six days. We identified sets of common and stimuli-specific genes, the expression of which changed following stimulation with LPS, TNF-alpha or CD40L. A dynamic analysis of the entire DC differentiation and maturation process showed that some important inflammatory and constitutive chemokines are transcribed in both immature and mature DC. The correlative expression kinetics of the gene pairs IL1R1/IL1R2, IL15/IL15RA, DC-SIGN/ICAM-2 and DC-SIGN/ICAM-3 imply that they all play crucial roles in mediating DC functions. Thus, our analysis with focused microarrays shed light on the transcriptional kinetics of DC differentiation and maturation, and this method may also prove useful for identifying novel marker genes involved in DC functions.